CN117616120A

CN117616120A - Variant lipases and uses thereof

Info

Publication number: CN117616120A
Application number: CN202280045956.6A
Authority: CN
Inventors: C·D·亚当斯; L·M·贝贝; A·D·刘; C·德格林; S·伊斯兰; S·维兰德
Original assignee: Danisco US Inc
Current assignee: Danisco US Inc
Priority date: 2021-06-30
Filing date: 2022-06-27
Publication date: 2024-02-27
Also published as: EP4363565A1; US20240294888A1; WO2023278297A1

Abstract

The present disclosure relates to variant lipolytic enzymes, more particularly to variant lipolytic enzymes having improved stability and/or improved hydrolytic activity towards polyesters. Such variant lipolytic enzymes may be used to degrade polyesters such as polyethylene terephthalate. Compositions and methods related to such variant lipolytic enzymes are also provided.

Description

Variant lipases and uses thereof

Cross reference

The present application claims the benefit of U.S. provisional application No. 63/216,612, filed on 6/30 of 2021, and is incorporated by reference in its entirety.

Background

Polyesters, such as polyethylene terephthalate (PET), are used in a wide variety of products and processes, such as in the manufacture of clothing, carpeting, various packaging, and plastics, such as automotive plastics, which lead to accumulation of polyesters in landfills and can be an ecological problem.

Various enzymes (e.g., lipolytic enzymes) are capable of catalyzing the hydrolysis of a wide variety of polymers, including polyesters. Some of these enzymes are being studied for many industrial applications, such as detergents for laundry and dish washing applications, degrading enzymes for treating biomass and food, biocatalysts in detoxification of environmental pollutants or biocatalysts for treating polyester fabrics in the textile industry. The use of such enzymes is particularly interesting for hydrolyzing polyesters such as PET.

There is a continuing need for lipolytic enzymes with improved activity and/or improved stability which can be used in compositions for treating fabrics and/or textiles and in methods for degrading polyesters.

Disclosure of Invention

In one embodiment, the present disclosure provides a variant lipolytic enzyme comprising an amino acid sequence having at least 70% identity to the full length amino acid sequence of SEQ ID NO. 2, the variant lipolytic enzyme comprising the substitutions T064V-T117L-T177N/R-I178L-F180P-Y182A-R190L-S205G-S212D-F226L-Y239I-L249P-S252I-L258F and further comprising at least one additional substitution selected from the group consisting of: V014S, R040A/T, G059Y, G061D, A066D, S070E, Q161H, G A/E, F207TL/T, V210I, Q227H, A236P, S244E, E Q and R256K, wherein these positions are numbered by reference to the amino acid sequence of SEQ ID NO:2 and wherein the variant has esterase activity. In some embodiments, the variant lipolytic enzyme comprises an amino acid sequence which is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the full length amino acid sequence of SEQ ID NO. 2. In some embodiments, the variant lipolytic enzyme is derived from a parent enzyme comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the full-length amino acid sequence of SEQ ID NO. 2.

In another embodiment, the present disclosure provides a polynucleotide comprising a nucleic acid sequence encoding a variant lipolytic enzyme comprising an amino acid sequence having at least 70% identity to the full length amino acid sequence of SEQ ID NO. 2, comprising the substitutions T064V-T117L-T177N/R-I178L-F180P-Y182A-R190L-S205G-S212D-F226L-Y239I-L249P-S252I-L258F, and further comprising at least one additional substitution selected from the group consisting of: V014S, R040A/T, G059Y, G061D, A066D, S070E, Q161H, G A/E, F207TL/T, V210I, Q227H, A236P, S244E, E Q and R256K, wherein these positions are numbered by reference to the amino acid sequence of SEQ ID NO:2 and wherein the variant has esterase activity. In some embodiments, the variant lipolytic enzyme comprises an amino acid sequence which is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the full length amino acid sequence of SEQ ID NO. 2. In some embodiments, the variant lipolytic enzyme is derived from a parent enzyme comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the full-length amino acid sequence of SEQ ID NO. 2.

In another embodiment, the present disclosure provides expression vectors or cassettes comprising polynucleotides encoding variant lipolytic enzymes, and recombinant host cells containing such expression vectors or cassettes.

Also provided are enzyme compositions comprising a variant lipolytic enzyme comprising an amino acid sequence having at least 70% identity to the full length amino acid sequence of SEQ ID NO. 2, comprising the substitutions T064V-T117L-T177N/R-I178L-F180P-Y182A-R190L-S205G-S212D-F226L-Y239I-L249P-S252I-L258F, and further comprising at least one additional substitution selected from the group consisting of: V014S, R040A/T, G059Y, G061D, A066D, S070E, Q161H, G A/E, F207TL/T, V210I, Q227H, A236P, S244E, E Q and R256K, wherein these positions are numbered by reference to the amino acid sequence of SEQ ID NO:2 and wherein the variant has esterase activity. In some embodiments, the variant lipolytic enzyme comprises an amino acid sequence which is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the full length amino acid sequence of SEQ ID NO. 2. In some embodiments, the variant lipolytic enzyme is derived from a parent enzyme comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the full-length amino acid sequence of SEQ ID NO. 2.

The present disclosure further provides methods for degrading polyesters or polyester-containing materials and methods for enzymatically depolymerizing polyesters or polyester-containing materials. Such a process may be used for polyesters selected from the group consisting of: polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), polyethylene naphthalate (PEN), polyester polyurethane, poly (ethylene adipate) (PEA), and combinations thereof.

Detailed Description

The present disclosure provides variant lipolytic enzymes, compositions (e.g., enzyme and detergent compositions) comprising such variant lipolytic enzymes, and methods of using such variant lipolytic enzymes and compositions, e.g., for washing or treating textiles and/or fabrics, and degrading polyesters.

Before describing embodiments of the compositions and methods of the present invention, the following terms are defined.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the specification in general. Furthermore, as used herein, the singular terms "a" and "an" and "the" include plural referents unless the context clearly dictates otherwise. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary depending upon the context in which they are used by those skilled in the art.

Every maximum numerical limitation given throughout this specification is intended to include every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

As used herein, the term "polymer" refers to a compound or mixture of compounds whose structure is made up of multiple repeating units linked by covalent chemical bonds. In the context of the present disclosure, the term polymer includes natural or synthetic polymers that are composed of a single type of repeating unit (i.e., homopolymers) or a mixture of different repeating units (i.e., block copolymers and random copolymers).

As used herein, the term "polyester-containing material" or "polyester-containing product" refers to a product, such as a textile, fabric, or plastic product, comprising at least one polyester in crystalline, semi-crystalline, or substantially amorphous form. In some embodiments, polyester-containing material refers to any article made of at least one plastic material, such as plastic sheets, plastic tubes, plastic rods, plastic profiles, plastic forms (plastic shapes), plastic films, plastic blocks (plastic blocks), etc., containing at least one polyester, and possibly other substances or additives, such as plasticizers, minerals, or organic fillers. In some embodiments, polyester-containing material refers to a plastic compound, or plastic formulation, in a molten or solid state, that is suitable for making a plastic product. In some embodiments, polyester-containing material refers to a textile or fabric or fiber comprising at least one polyester. In some embodiments, polyester-containing material refers to plastic waste or fiber waste comprising at least one polyester.

As used herein, the term "polyester" refers to monomers of which are bonded by ester linkages. As used herein, the term "polyester" includes, but is not limited to, those polyesters selected from the group consisting of: polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), polyethylene naphthalate (PEN), polyester polyurethane, poly (ethylene adipate) (PEA), and combinations thereof.

The term "fabric" refers to, for example, woven, knitted, and non-woven materials, as well as staple fibers and filaments that can be converted into, for example, yarns and woven, knitted, and non-woven materials. The term encompasses materials made from natural as well as synthetic (e.g., manufactured) fibers, and combinations thereof.

As used herein, the term "textile" refers to any textile material, including yarns, yarn intermediates, fibers, nonwoven materials, natural materials, synthetic materials, and any other textile material, fabrics made from such materials, and products made from fabrics (e.g., garments and other articles). The textile or fabric may be in the form of a knit, woven, jean, nonwoven, felt, yarn, and terry cloth. The textile may comprise a cellulose-based, such as a natural cellulosic product, including cotton, flax/linen, jute, ramie, sisal, or coir, or a man-made cellulose (e.g., derived from wood pulp), including viscose/rayon, cellulose acetate (tricell), lyocell, or blends thereof. The textile or fabric may also be non-cellulose based, such as natural polyamides including wool, camel hair, cashmere, mohair, rabbit hair and silk, or synthetic polymers such as nylon, aramid, polyester, acrylic, polypropylene and spandex/elastane (spandex/elastane), or blends thereof and blends of cellulose-based and non-cellulose-based fibers. Examples of blends are blends of cotton and/or rayon/viscose with one or more companion materials (companion material) such as wool, synthetic fibers (e.g., polyamide fibers, acrylic fibers, polyester fibers, polyvinyl chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers) and/or cellulose-containing fibers (e.g., rayon/viscose, ramie, flax/linen, jute, cellulose acetate fibers, lyocell fibers). The fabric may be a conventional washable garment, such as stained household garments. When the term fabric or garment is used, the broad term textile is intended to be included as well. In the context of this application, the term "textile" is used interchangeably with fabric and cloth. In some embodiments, the textile comprises those materials comprising at least one polyester.

The term "washing" includes both home washing and industrial washing and means the process of treating a textile with a solution containing a cleaning or detergent composition as provided herein. The washing process may be performed, for example, using a household or industrial washing machine, or may be performed by hand.

The term "wash cycle" refers to a washing operation in which the textile is immersed in a wash liquor, some mechanical action is applied to the textile to release stains or to facilitate the flow of wash liquor into and out of the textile, and finally excess wash liquor is removed. After one or more wash cycles, the textiles are typically rinsed and dried.

The term "wash liquor" is defined herein as a solution or mixture of water and detergent components, optionally including a variant lipolytic enzyme as provided herein.

As used herein, "homologous genes" refers to pairs of genes from different, but generally related, species that correspond to each other and are identical or very similar to each other. The term encompasses genes isolated by speciation (i.e., development of a new species) (e.g., orthologous genes) as well as genes isolated by genetic duplication (e.g., paralogous genes).

As used herein, the term "variant polypeptide" refers to a polypeptide comprising an amino acid sequence that differs from the amino acid sequence of a parent polypeptide or reference polypeptide (including but not limited to wild-type polypeptides) by at least one amino acid residue. In some embodiments, a parent polypeptide for use herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 2.

Variant lipolytic enzymes

In one embodiment, a variant lipolytic enzyme is provided. In some embodiments, the variant lipolytic enzymes provided herein have hydrolytic activity on at least one polyester.

As used herein, lipolytic enzymes include enzymes, polypeptides, or proteins that exhibit the ability to degrade lipids (e.g., the ability to degrade triglycerides or phospholipids). The lipolytic enzyme may be, for example, a lipase, phospholipase, esterase or cutinase. The lipolytic enzyme may be an enzyme having an alpha/beta hydrolase folding. These enzymes typically have catalytic triplets of serine, aspartic acid and histidine residues. Alpha/beta hydrolases include lipases and cutinases. Cutinases exhibit little, if any, interfacial activation, with lipases typically undergoing conformational changes in the presence of a lipid-water interface (Longhi and cambrilla u (1999) Biochimica et Biophysica Acta [ journal of biochemistry and biophysics ] 1441:185-96). The active fragment of a lipolytic enzyme is the part of the lipolytic enzyme that retains the ability to degrade lipids. The active fragment retains the catalytic triad. As used herein, lipolytic activity may be determined according to any procedure known in the art (see, e.g., gupta et al, biotechnol. Appl. Biochem. [ biotech applications and biochemistry ],37:63-71,2003; U.S. Pat. No. 5,990,069; and international patent publication No. WO 96/18729 A1).

In some embodiments, the lipolytic enzyme of the present disclosure is an alpha/beta hydrolase. In some embodiments, the lipolytic enzyme of the present disclosure is a lipase. In some embodiments, the lipolytic enzyme of the present disclosure is a cutinase. In some embodiments, the lipolytic enzyme of the present disclosure is an esterase.

In some embodiments, the lipolytic enzyme of the present disclosure is an alpha/beta hydrolase. In some embodiments, the lipolytic enzyme of the present disclosure is a lipase. In some embodiments, the lipolytic enzyme of the present disclosure is a cutinase. In some embodiments, the lipolytic enzyme of the present disclosure is a polyesterase.

As used herein, "carboxylate hydrolase" (e.c. 3.1.1) refers to an enzyme that acts on a carboxylate ester.

As used herein, "lipase, lipase enzyme," lipolytic enzyme, "lipolytic polypeptide," or "lipolytic protein" refers to an enzyme, polypeptide, or protein that exhibits the ability to degrade lipids (e.g., the ability to degrade triglycerides or phospholipids). The lipolytic enzyme may be, for example, a lipase, phospholipase, esterase, polyesterase, or cutinase. As used herein, lipolytic activity may be determined according to any procedure known in the art (see, e.g., gupta et al, biotechnol. Appl. Biochem. [ biotech applications and biochemistry ],37:63-71,2003; U.S. Pat. No. 5,990,069; and international patent publication No. WO 96/18729 A1). In one example, lipolytic activity may be determined on 4-nitrophenyl butyrate (pNB) as provided in example 2.

As used herein, "cutinase" refers to a lipolytic enzyme capable of hydrolyzing a cutin substrate.

Cutinases include those derived from a variety of fungal and bacterial sources. Cutinases include those described below: kolattukudy, "Lipases [ Lipase ]", B Borgstrom and H.L. Brockman editions, elsevier [ Escule Press ]1984,471-504; s.longhi et al, J.of Molecular Biology [ journal of molecular biology ],268 (4), 779-799 (1997); U.S. Pat. nos. 5,827,719; WO 94/14963; WO 94/14964; WO 00/05389; appl.environm.microbiol [ application and environmental microbiology ]64,2794-2799,1998; protein: structure, function and Genetics [ protein: structure, function, and genetics 26,442-458,1996; j.of Computational Chemistry [ journal of computational chemistry ]17,1783-1803,1996; protein Engineering [ protein engineering ]6,157-165,1993. The cutinase may be a naturally occurring cutinase or a genetically modified cutinase obtained by UV irradiation, N-methyl-N' -Nitrosoguanidine (NTG) treatment, ethyl Methanesulfonate (EMS) treatment, nitrous acid treatment, acridine treatment, or the like, recombinant strains induced by genetic engineering procedures (e.g., cell fusion, gene recombination, and the like).

As used herein, the term "polyesterase" or "PET enzyme" refers to an enzyme that has a significant ability to catalyze hydrolysis and/or surface modification of polyesters. Suitable polyesterase enzymes can be isolated from animal, plant, fungal and bacterial sources. In addition to isolation from wild strains, the aforementioned microorganisms may be isolated from any mutant strains obtained by UV irradiation, N-methyl-N' -Nitrosoguanidine (NTG) treatment, ethyl Methanesulfonate (EMS) treatment, nitrous acid treatment, acridine treatment, etc., recombinant strains induced by genetic engineering procedures such as cell fusion and gene recombination, etc. The polyesterase may catalyze the hydrolysis and/or surface modification of polyesters selected from the group consisting of: polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), polyethylene naphthalate (PEN), polyester polyurethane, poly (ethylene adipate) (PEA), and combinations thereof.

As used herein, "% identity or percent identity" refers to sequence similarity. The percent identity can be determined using standard techniques known in the art (see, e.g., smith and Waterman, adv. Appl. Math. [ applied math. Progress ]2:482[1981], needleman and Wunsch, J. Mol. Biol. [ J. Mol. Biol. ]48:443[1970], pearson and Lipman, proc. Natl. Acad. Sci. USA [ Proc. Sci. U.S. Sci. ]85:2444[1988]; software programs in the genetics computer group (Genetics Computer Group, madison, wis.) of Madison, wis., such as GAP, BESTFIT, FASTA and TFASTA; devereux et al, nucl acid Res. [ nucleic acids research ]12:387-395[1984 ]). One example of a useful algorithm is PILEUP. PILEUP creates multiple sequence alignments from a set of related sequences using progressive, pairwise alignments. It may also plot and display a tree of the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (see Feng and Doolittle, J.mol. Evol. [ J. Molecular evolution ]35:351-360[1987 ]). This method is similar to that described by Higgins and Sharp (see Higgins and Sharp, CAWIOS [ computer applications in bioscience ]5:151-153[1989 ]). Useful PILEUP parameters include a default slot weight of 3.00, a default slot length weight of 0.10, and a weighted end slot. Other useful algorithms are the BLAST algorithm described by Altschul et al (see Altschul et al, J.mol. Biol. [ J. Mol. Biol. Mol. 215:403-410[1990 ]) Karlin and Altschul, proc. Natl. Acad. Sci. USA [ Proc. Sci. Natl. Acad. Sci ]90:5873-5787[1993 ]). The BLAST program uses several search parameters, most of which are set to default values.

As used herein, "homologous protein," "homolog," or "homologous protein" refers to a protein that has substantial similarity in primary, secondary, and/or tertiary structure. When proteins are aligned, protein homology may refer to the similarity of linear amino acid sequences. Homology can be determined by amino acid sequence alignment, for example using programs such as BLAST, MUSCLE or CLUSTAL. Homology searches for protein sequences can be performed using BLASTP and PSI-BLAST from NCBI BLAST using a threshold value of 0.001 (E value cutoff). (Altschul et al, "Gapped BLAST and PSI BLAST a new generation of protein database search programs" [ vacancy BLAST and PSI BLAST: new generation protein database search program ], nucleic Acids Res [ nucleic acids research ], group 1; 25 (17): 3389-402 (1997)). The BLAST program uses several search parameters, most of which are set to default values. The NCBI BLAST algorithm finds the most relevant sequences according to biological similarity, but is not recommended for query sequences of less than 20 residues (Altschul et al, nucleic Acids Res [ nucleic acids research ],25:3389-3402,1997 and Schaffer et al, nucleic Acids Res [ nucleic acids research ],29:2994-3005,2001). Exemplary default BLAST parameters for nucleic acid sequence searches include: adjacent word length threshold = 11; e value cutoff = 10; scoring Matrix (Scoring Matrix) =nuc.3.1 (match=1, mismatch= -3); vacancy open = 5; and vacancy extension = 2. Exemplary default BLAST parameters for amino acid sequence searches include: word length = 3; e value cutoff = 10; score matrix = BLOSUM62; vacancy open = 11; and vacancy extension = 1. Using this information, protein sequences can be grouped and/or phylogenetic trees constructed therefrom. Amino acid sequences can be entered in programs such as the Vector NTI Advance suite, and guide trees can be created using the adjacency (NJ) method (Saitou and Nei, mol Biol Evol [ molecular biology and evolution ],4:406-425,1987). The tree structure can be calculated using Kimura correction for sequence distance and ignoring positions with gaps. A program such as AlignX may display the calculated distance values in brackets after the molecular names displayed on the phylogenetic tree.

Percent (%) amino acid sequence identity values are determined by dividing the number of matching identical residues by the total number of residues of the "reference" sequence (including any gaps created by the program for optimal/maximum alignment). SEQ ID NO: A is a "reference" sequence if the sequence is 90% identical to SEQ ID NO: A. The BLAST algorithm refers to the "reference" sequence as a "query" sequence.

The CLUSTAL W algorithm is another example of a sequence alignment algorithm (see Thompson et al, nucleic Acids Res [ nucleic acids Ind. 22:4673-4680,1994). Default parameters for the CLUSTAL W algorithm include: gap opening penalty = 10.0; gap extension penalty = 0.05; protein weight matrix = BLOSUM series; DNA weight matrix = IUB; delay divergent sequence% = 40; gap separation distance = 8; DNA conversion weight = 0.50; list hydrophilic residues = GPSNDQEKR; using negative matrix = off; switching special residue penalty = on; switch hydrophilic penalty = on; and switching end gap separation penalty = off. Deletions occurring at either end are included in the CLUSTAL algorithm. For example, a variant having five amino acid deletions at either end of a 500 amino acid polypeptide (or within a polypeptide) has a percent sequence identity of 99% (495/500 identical residues x 100) relative to a "reference" polypeptide. Such variants will be encompassed by variants having "at least 99% sequence identity" to the polypeptide.

In some embodiments, variant lipases include those derived from 2FX5_A, as well as those derived from lipases disclosed in WO88/09367, U.S. Pat. No. 5,512,203, 5,389,536, U.S. Pat. publication No. US2003199068, european patent publication No. EP1543117, and WO 03/076580.

In some embodiments, variant lipolytic enzymes provided herein comprise an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO. 2. In some embodiments, the variant lipolytic enzyme has an amino acid sequence which has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID No. 2 and has esterase activity.

The present disclosure provides variant lipolytic enzymes or active fragments thereof comprising an amino acid sequence having at least 70% identity to the full length amino acid sequence of SEQ ID NO. 2, comprising the substitutions T064V-T117L-T177N/R-I178L-F180P-Y182A-R190L-S205G-S212D-F226L-Y239I-L249P-S252I-L258F, and further comprising at least one additional substitution selected from the group consisting of: V014S, R040A/T, G059Y, G061D, A066D, S070E, Q161H, G A/E, F207TL/T, V210I, Q227H, A236P, S244E, E Q and R256K, wherein these positions are numbered by reference to the amino acid sequence of SEQ ID NO:2 and wherein the variant has esterase activity.

In some embodiments, the variant lipolytic enzyme provided herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the full-length amino acid sequence of SEQ ID NO. 2, which comprises the substitution T064V-T117L-T177N/R-I178L-F180P-Y182A-R190L-S205G-S212D-F226L-Y239I-L249P-S252I-L258F and further comprises at least one additional substitution selected from the group consisting of: V014S, R040A/T, G059Y, G061D, A066D, S070E, Q161H, G A/E, F207TL/T, V210I, Q227H, A236P, S244E, E Q and R256K, wherein these positions are numbered by reference to the amino acid sequence of SEQ ID NO:2 and wherein the variant has esterase activity.

In some embodiments, the variant lipolytic enzyme provided herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the full-length amino acid sequence of SEQ ID NO:2 and comprises a combination of mutations selected from the group consisting of: R40T-T64V-T117T-G175L-T180G-G175A-R190G-S205G-F207L-S212D-F226L-Y239I-L249P-S252I-L258F, R T-G61D-T64V-S70E-T117L-T177L-S178L-F180P-Y180A-R190L-S205G-F207T-S212D-F226L-Q227H-A236P-Y239I-L249P-S252I-E258Q-L F, R T64T-T64V-S70E-T117L-T117N-I178L-F182A-R182L-S205T-F207T-S212D-F226L-A236P-Y249P-S252I-E254Q-L5740A-T64V-E70T-T180F 180L-T180I-S182A-L182-D180F-L180; F226L-A236P-Y239I-L249P-S252I-E254Q-L258F, R T-T64V-S70E-T117L-Q161H-T177N-I178L-F180P-Y182A-R190L-S205G-F207T-S212D-F226L-A236P-Y239I-L249P-S252I-E254Q-L258F, R T-T64V-S70E-T117L-G117A-T177N-I178L 180P-Y182A-R190L-S205G-F207T-S212D-F226L-A236P-Y239I-L249P-S252P-E254Q-L258F, R T64V-S70E-T117L-T177N-I178L 180P-Y182A-R190L-S190G-S205T 210F 180P-E252A-F252D 252L-E258P-E252L-E252L-I254, R40T-T64V-T70E-T117L-T178L-F180P-Y182A-R190L-S205G-F207T-S212D-F226L-A236P-Y239I-S244E-L249P-S254I-E254Q-L258T 40T-T64V-S70E-T117L-T177N-I178L-F180P-Y182A-R190L-S205G-F207T-S212D-F226L-A236P-Y249P-S254Q-R256K-L258F, V A-G61Y-G61D-T64V-A66D-S70E-T117L-Q161H-T177R-I178L-F180P-Y180A-R190L-S205G-F207T-S210I-S212D-F226L-A236P-Y239I-L249P-S252I-E254Q-R256K-E254K-T64V-S70E-T117S-R40A-G59Y-G61D-T64V-S70E-R190L-S205G-F207T-V210I-S212D-F226L-A236P-Y239I-L249P-S252I-E254Q-R256K-L258 6240T-G61D-T64V-S70E-T117L-Q161H-T177R-178L-F180P-Y182A-R205G-F205T-V210I-S212D-F226L-A236P-Y239I-L249P-S252I-E254Q-R256K-L258F 258, and V14S-R40A-G59Y-G61D-T64V-A66D-S70E-T117L-Q161H-G175A-T177R-I178L-F180P-Y182A-R190L-S205G-F207T-V210I-S212D-F226L-A236P-Y239I-L249P-S252I-E254Q-R256K-L258F, wherein these positions are numbered by reference to the amino acid sequence of SEQ ID NO: 2.

In some embodiments, a variant lipolytic enzyme provided herein has esterase activity (e.g., the ability to catalyze hydrolysis and/or surface modification) on at least one polyester selected from the group consisting of: polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), polyethylene naphthalate (PEN), polyester polyurethane, poly (ethylene adipate) (PEA), and combinations thereof. In one embodiment, the variant lipolytic enzyme provided herein has esterase activity on PET.

Described herein are one or more isolated, non-naturally occurring, or recombinant polynucleotides comprising a nucleic acid sequence encoding one or more variant lipolytic enzymes described herein, or a recombinant polypeptide or an active fragment thereof. The one or more nucleic acid sequences described herein may be used in the recombinant production (e.g., expression) of one or more variant lipolytic enzymes described herein, typically by expression of a plasmid expression vector comprising a sequence encoding one or more variant lipolytic enzymes described herein or fragments thereof. One embodiment provides a nucleic acid encoding one or more variant lipolytic enzymes described herein, wherein the variant is a mature form having lipolytic activity. In some embodiments, one or more variant lipolytic enzymes described herein are recombinantly expressed with a homologous propeptide sequence. In other embodiments, one or more variant lipolytic enzymes described herein are recombinantly expressed with a heterologous propeptide sequence.

One or more of the nucleic acid sequences described herein may be produced using any suitable synthesis, manipulation, and/or isolation technique, or combination thereof. For example, one or more polynucleotides described herein may be produced using standard nucleic acid synthesis techniques, such as solid phase synthesis techniques, well known to those of skill in the art. In such techniques, fragments of up to 50 or more nucleotide bases are typically synthesized and then ligated (e.g., by enzymatic or chemical ligation methods) to form essentially any desired contiguous nucleic acid sequence. Synthesis of one or more polynucleotides described herein may also be facilitated by any suitable method known in the art, including, but not limited to, chemical synthesis using: classical phosphoramidite methods (see, e.g., beaucage et al, tetrahedron Letters [ tetrahedral flash ]22:1859-69 (1981)), or methods described in Matthes et al, EMBO J. [ J. European molecular biology J ]3:801-805 (1984), as typically practiced in automated synthesis methods. One or more polynucleotides described herein may also be produced by using an automated DNA synthesizer. Can be obtained from various commercial sources (e.g., ATUM (DNA 2.0), newwar (New, calif., USA), life technologies (Life technologies) (GeneArt), calif., bard (Carlsbad, calif., USA), kingrui (GenScript), ontario, canada, base Clear (Base Clear B.V.), netherlands (Leiden, netherlands), integrated DNA technologies (Integrated DNA Technologies), illinois (Skokie, IL, USA), ginkgo biological studio (Ginkgo Bioworks) (Gen 9), massachusetts (Boston, mass., USA), tex biosciences (Bioscience), twok Bioscience, calif., san France, USA). Other techniques and related principles for synthesizing nucleic acids are described, for example, by Itakura et al, ann.Rev.biochem. [ Biochemical annual. 53:323 (1984) and Itakura et al, science [ Science ]198:1056 (1984).

Recombinant DNA techniques for modifying nucleic acids are well known in the art, such as, for example, restriction endonuclease digestion, ligation, reverse transcription and cDNA production, and polymerase chain reaction (e.g., PCR). One or more polynucleotides described herein may also be obtained by screening a cDNA library using one or more oligonucleotide probes that can hybridize to or PCR amplify a polynucleotide encoding one or more variant lipolytic enzymes, or recombinant polypeptides, or active fragments thereof, described herein. Procedures for screening and isolating cDNA clones and PCR amplification procedures are well known to those skilled in the art and are described in standard references known to those skilled in the art. One or more polynucleotides described herein can be obtained, for example, by altering a naturally occurring polynucleotide backbone (e.g., a polynucleotide backbone encoding one or more variant lipolytic enzymes or reference lipolytic enzymes described herein) by known mutagenesis procedures (e.g., site-directed mutagenesis, site-saturation mutagenesis, and in vitro recombination). Various methods suitable for producing modified polynucleotides described herein that encode one or more variant lipolytic enzymes described herein are known in the art, including, but not limited to, for example, site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, deletion mutagenesis, random mutagenesis, site-directed mutagenesis and directed evolution, as well as various other recombinant methods.

Further embodiments relate to one or more vectors comprising one or more variant lipolytic enzymes described herein (e.g., polynucleotides encoding one or more variant lipolytic enzymes described herein); expression vectors or expression cassettes comprising one or more nucleic acid or polynucleotide sequences described herein; isolated, substantially pure, or recombinant DNA constructs comprising one or more nucleic acid or polynucleotide sequences described herein; an isolated or recombinant cell comprising one or more polynucleotide sequences described herein; and compositions comprising one or more such vectors, nucleic acids, expression vectors, expression cassettes, DNA constructs, cells, cell cultures, or any combination or mixture thereof.

Some embodiments relate to one or more recombinant cells comprising one or more vectors (e.g., expression vectors or DNA constructs) described herein comprising one or more nucleic acid or polynucleotide sequences described herein. Some such recombinant cells are transformed or transfected with such at least one vector, although other methods are available and known in the art. Such cells are typically referred to as host cells. Some such cells include bacterial cells, including but not limited to Bacillus species cells, such as Bacillus subtilis cells. Other embodiments relate to recombinant cells (e.g., recombinant host cells) comprising one or more variant lipolytic enzymes described herein.

In some embodiments, one or more vectors described herein are expression vectors or expression cassettes comprising one or more polynucleotide sequences described herein operably linked to one or more additional nucleic acid segments (e.g., a promoter operably linked to one or more polynucleotide sequences described herein) required for efficient gene expression. The vector may include a transcription terminator and/or a selection gene (e.g., an antibiotic resistance gene) capable of achieving continuous culture maintenance of the plasmid-infected host cell by growth in a medium containing the antimicrobial agent.

The expression vector may be derived from plasmid or viral DNA, or in alternative embodiments, contain elements of both. Exemplary vectors include, but are not limited to, pC194, pJH101, pE194, pHP13 (see Harwood and Cutting [ eds. ], chapter 3, molecular Biological Methods for Bacillus [ methods of molecular biology for Bacillus ], john Wiley & Sons [ John Willi parent ] (1990); suitable replicators for Bacillus subtilis include those listed on page 92). (see also, perego, "Integrational Vectors for Genetic Manipulations in Bacillus subtilis [ integrative vector for genetic manipulation in Bacillus subtilis ]"; sonenshein et al, [ edit ]; "Bacillus subtilis and Other Gram-Positive Bacteria: biochemistry, physiology and Molecular Genetics [ Bacillus subtilis and other gram positive bacteria: biochemistry, physiology and molecular genetics ]", american Society for Microbiology [ American society of microbiology ], washington, D.C. [ Columbia, washington ] (1993), pages 615-624, and p2JM103 BBI).

To express and produce a protein of interest (e.g., one or more variant lipolytic enzymes described herein) in a cell, one or more expression vectors comprising one or more copies (and in some cases, multiple copies) of a polynucleotide encoding one or more variant lipolytic enzymes described herein are transformed into the cell under conditions suitable for expression of the variant. In some embodiments, polynucleotide sequences encoding one or more variant lipolytic enzymes described herein (as well as other sequences contained in a vector) are integrated into the genome of a host cell; in other embodiments, however, a plasmid vector comprising a polynucleotide sequence encoding one or more variant lipolytic enzymes described herein remains as an autonomous extrachromosomal element within the cell. Some embodiments provide an extrachromosomal nucleic acid element and an import nucleotide sequence integrated into the host cell genome. The vectors described herein can be used to produce one or more variant lipolytic enzymes described herein. In some embodiments, the polynucleotide construct encoding one or more variant lipolytic enzymes described herein is present on an integrating vector capable of integrating the polynucleotide encoding the variant into a host chromosome and optionally amplifying in the host chromosome. Examples of integration sites are well known to those skilled in the art. In some embodiments, transcription of a polynucleotide encoding one or more variant lipolytic enzymes described herein is effected by a promoter that is the wild-type promoter of the parent enzyme. In some other embodiments, the promoter is heterologous to one or more variant lipolytic enzymes described herein, but is functional in the host cell. Exemplary promoters for bacterial host cells include, but are not limited to, the amyE, amyQ, amyL, pstS, sacB, pSPAC, pAprE, pVeg, pHpaII promoter; a promoter of the Bacillus stearothermophilus maltogenic amylase gene; a promoter of the Bacillus Amyloliquefaciens (BAN) amylase gene; a promoter of the bacillus subtilis alkaline protease gene; a promoter of the alkaline protease gene of bacillus clausii (b.clausii); a promoter of the Bacillus pumilus (B.pumilis) xylosidase gene; the promoter of bacillus thuringiensis (b. Thuringiensis) cryIIIA; and the promoter of the Bacillus licheniformis (B.lichenifermis) alpha-amylase gene. Additional promoters include, but are not limited to, the A4 promoter, and the phage λPR or PL promoters, as well as the E.coli (E.coli) lac, trp or tac promoters.

The one or more variant lipolytic enzymes described herein may be produced in a host cell of any suitable microorganism, including bacteria and fungi. In some embodiments, one or more variant lipolytic enzymes described herein may be produced in gram-positive bacteria. In some embodiments, the host cell is a Bacillus species, streptomyces (Streptomyces) species, escherichia (Escherichia) species, aspergillus (Aspergillus) species, trichoderma (Trichoderma) species, pseudomonas (Pseudomonas) species, corynebacterium (Corynebacterium) species, saccharomyces (Saccharomyces) species, or Pichia (Pichia) species. In some embodiments, one or more variant lipolytic enzymes described herein are produced by a bacillus species host cell. Examples of bacillus species host cells that can be used for the production of one or more variant lipolytic enzymes described herein include, but are not limited to: bacillus licheniformis, bacillus lentus (B.lentus), bacillus subtilis, bacillus amyloliquefaciens, bacillus brevis (B.brevis), bacillus stearothermophilus, bacillus alcalophilus (B.allophilus), bacillus coagulans (B.coagulans), bacillus circulans (B.circulans), bacillus pumilus, bacillus thuringiensis, bacillus clausii and Bacillus megaterium (B.megaterium), among other organisms within the genus Bacillus. In some embodiments, a bacillus subtilis host cell is used to produce the variants described herein. USPN 5,264,366 and 4,760,025 (RE 34,606) describe various bacillus host strains that can be used to produce one or more variant lipolytic enzymes described herein, although other suitable strains can be used.

Several bacterial strains that can be used to produce one or more variant lipolytic enzymes described herein include non-recombinant (i.e., wild-type) strains of bacillus species, as well as naturally occurring strains and/or variants of recombinant strains. In some embodiments, the host strain is a recombinant strain in which a polynucleotide encoding one or more variant lipolytic enzymes described herein has been introduced into the host. In some embodiments, the host strain is a bacillus subtilis host strain, in particular a recombinant bacillus subtilis host strain. Many strains of Bacillus subtilis are known, including but not limited to, for example, 1A6 (ATCC 39085), 168 (1A 01), SB19, W23, ts85, B637, PB1753 through PB1758, PB3360, JH642, 1A243 (ATCC 39,087), ATCC 21332, ATCC 6051, MI113, DE100 (ATCC 39,094), GX4931, PBT 110, and PEP 211 strains (see, e.g., hoch et al, genetics [ Genetics ]73:215-228 (1973); see also U.S. Pat. No. 3,284; and EP 01334048). The use of Bacillus subtilis as an expression host cell is well known in the art (see, e.g., palva et al, gene [ Gene ]19:81-87 (1982); fahnestock and Fischer, J. Bacteriol. [ J. Bacteriol., 165:796-804 (1986); and Wang et al, gene [ Gene ]69:39-47 (1988)).

In some embodiments, the bacillus host cell is a bacillus species comprising a mutation or deletion in at least one of the following genes: degU, degS, degR and degQ. In some embodiments, the mutation is in the degU gene, and in some embodiments, the mutation is degU (Hy) 32 (see, e.g., msadek et al, J. Bacteriol. [ J. Bacteriol. ]172:824-834 (1990); and Olmos et al, mol. Gen. Genet. [ molecular and general genetics ]253:562-567 (1997)). In some embodiments, the bacillus host comprises a mutation or deletion in: scoC4 (see, e.g., caldwell et al, J.bacteriol. [ J.bacteriology ]183:7329-7340 (2001)); spoIIE (see, e.g., arigoni et al, mol. Microbiol. [ molecular microbiology ]31:1407-1415 (1999)); and/or other genes of the oppA or opp operon (see, e.g., perego et al, mol. Microbiol. [ molecular microbiology ]5:173-185 (1991)). Indeed, it is contemplated that any mutation in the opp operon that causes the same phenotype as the mutation in the oppA gene will be useful in some embodiments of the altered bacillus strains described herein. In some embodiments, these mutations occur alone, while in other embodiments, a combination of mutations is present. In some embodiments, the altered bacillus host cell strain that can be used to produce one or more variant lipolytic enzymes described herein is a bacillus host strain that already comprises mutations in one or more of the genes described above. In addition, bacillus species host cells comprising one or more mutations and/or one or more deletions of endogenous protease genes may be used. In some embodiments, the bacillus host cell comprises a deletion of the aprE and nprE genes. In other embodiments, the Bacillus species host cell comprises a deletion of 5 protease genes, while in other embodiments, the Bacillus species host cell comprises a deletion of 9 protease genes (see, e.g., US 2005/0202535).

The host cell is transformed with one or more nucleic acid sequences encoding one or more variant lipolytic enzymes described herein using any suitable method known in the art. Methods for introducing nucleic acids (e.g., DNA) into bacillus cells or e.coli cells using plasmid DNA constructs or vectors and transforming such plasmid DNA constructs or vectors into such cells are well known. In some embodiments, the plasmid is then isolated from an E.coli cell and transformed into a Bacillus cell. However, the use of an intervening microorganism such as E.coli is not necessary, and in some embodiments, the DNA construct or vector is introduced directly into the Bacillus host.

Exemplary methods of introducing one or more nucleic acid sequences described herein into a Bacillus cell are described, for example, in Ferrari et al, "Genetics [ Genetics ]", in Hardwood et al [ edit ], bacillus [ Bacillus ], plenum Publishing Corp [ Protein publishing company ] (1989), pages 57-72; saunders et al, J.Bacteriol. [ J.Bacteriol., 157:718-726 (1984); hoch et al, J.Bacteriol. [ journal of bacteriology ],93:1925-1937 (1967); mann et al, current Microbiol [ modern microbiology ],13:131-135 (1986); holubova, folia Microbiol. [ Furilian microbiology ],30:97 (1985); chang et al mol. Gen. Genet. [ molecular and general genetics ]168:11-115 (1979); vorobjeva et al, FEMS microbiol. Lett. [ FEMS microbiology letters ]7:261-263 (1980); smith et al, appl.env.Microbiol [ application and environmental microorganisms ]51:634 (1986); fisher et al, arch. Microbiol. [ microbiology archives ],139:213-217 (1981); mcDonald, J.Gen.Microbiol [ journal of genetic microbiology ]130:203 (1984). Indeed, methods such as transformation (including protoplast transformation and transfection, transduction, and protoplast fusion) are well known and suitable for use herein. Methods known in the art for transforming bacillus cells include, for example, methods such as Plasmid marker rescue transformation, which involve uptake of donor plasmids by competent cells carrying partially homologous resident plasmids (see, contente et al, plasmid [ Plasmid ]2:555-571 (1979); haima et al, mol. Gen. Genet. [ molecular and general genetics ]223:185-191 (1990); weinaruch et al, J. Bacteriol.; 154:1077-1087 (1983); and Weinaruch et al, J. Bacteriol.; 169:1205-1211 (1987)). In this method, the input donor plasmid recombines with the homologous region of the resident "helper" plasmid during the process of mimicking chromosomal transformation.

In addition to the methods commonly used, in some embodiments, the host cell is directly transformed with a DNA construct or vector comprising a nucleic acid encoding one or more variant lipolytic enzymes described herein (i.e., the DNA construct or vector is not amplified or otherwise processed using intermediate cells prior to introduction into the host cell). Introduction of the DNA constructs or vectors described herein into a host cell includes those physical and chemical methods known in the art for introducing nucleic acid sequences (e.g., DNA sequences) into a host cell without insertion into the host genome. Such methods include, but are not limited to, calcium chloride precipitation, electroporation, naked DNA, and liposomes. In further embodiments, the DNA construct or vector is co-transformed with the plasmid without insertion of the plasmid. In further examples, the selectable marker is deleted from the altered Bacillus strain by methods known in the art (see, stahl et al J. Bacteriol. J. Bacterio. J. 158:411-418 (1984); and Palmeros et al Gene [ Gene ] 247:255-264 (2000)).

In some embodiments, the transformed cells are cultured in conventional nutrient media. Suitable specific culture conditions, such as temperature, pH, etc., are known to those skilled in the art and are described in detail in the scientific literature. Some embodiments provide cultures (e.g., cell cultures) comprising one or more of the variant lipolytic enzymes or nucleic acid sequences described herein.

In some embodiments, host cells transformed with one or more polynucleotide sequences encoding one or more variant lipolytic enzymes described herein are cultured in a suitable nutrient medium under conditions allowing expression of the variants, after which the resulting variants are recovered from the culture. In some embodiments, the variants produced by the cells are recovered from the culture medium by conventional procedures including, but not limited to, separation of the host cells from the culture medium, e.g., by centrifugation or filtration, precipitation of the protein component of the supernatant or filtrate by means of salts (e.g., ammonium sulfate), and chromatographic purification (e.g., ion exchange, gel filtration, affinity, etc.).

In some embodiments, one or more variant lipolytic enzymes produced by the recombinant host cell are secreted into the medium. Nucleic acid sequences encoding purification-promoting domains can be used to promote purification of the variants. The vector or DNA construct comprising a polynucleotide sequence encoding one or more variant lipolytic enzymes described herein may further comprise a nucleic acid sequence encoding a purification-promoting domain that promotes purification of the variant (see, e.g., kroll et al, DNA Cell Biol [ DNA Cell biology ]12:441-53 (1993)). Such purification-promoting domains include, but are not limited to, for example, metal chelating peptides, such as histidine-tryptophan modules that allow purification on immobilized metals (see Porath, protein expr. Purif. [ Protein expression and purification ]3:263-281[1992 ]), protein A domains that allow purification on immobilized immunoglobulins, and domains employed in FLAGS extension/affinity purification systems. It has also been found that the inclusion of cleavable linker sequences such as factor XA or enterokinase (e.g., sequences available from Invitrogen, san diego, california) between the purification domain and the heterologous protein can be used to facilitate purification.

Various methods can be used to determine the production level of one or more mature variant lipolytic enzymes described herein in a host cell. Such methods include, but are not limited to, methods such as using polyclonal or monoclonal antibodies specific for the enzyme. Exemplary methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence Immunoassay (FIA), and Fluorescence Activated Cell Sorting (FACS). These and other assays are well known in the art (see, e.g., maddox et al, J. Exp. Med. [ journal of laboratory medicine ]158:1211 (1983)). In another embodiment, methods that may be used include the assays provided in examples 2 and 3.

Some other embodiments provide methods for preparing or producing one or more mature variant lipolytic enzymes described herein. Mature variants do not include signal peptide or propeptide sequences. Some methods include preparing or producing one or more variant lipolytic enzymes described herein in a recombinant bacterial host cell, such as, for example, a bacillus species cell (e.g., a bacillus subtilis cell). Other embodiments provide methods of producing one or more variants described herein, wherein the method comprises culturing a recombinant host cell comprising a recombinant expression vector comprising a nucleic acid sequence encoding one or more variant lipolytic enzymes described herein under conditions conducive to production of the variant. Some such methods further comprise recovering the variant from the culture.

Further embodiments provide methods of producing one or more variant lipolytic enzymes described herein, wherein the methods comprise: (a) Introducing a recombinant expression vector comprising a nucleic acid encoding the variant into a population of cells (e.g., bacterial cells, such as bacillus subtilis cells); and (b) culturing the cells in a culture medium under conditions conducive to the production of the variant encoded by the expression vector. Some such methods further comprise: (c) isolating the variant from the cells or from the culture medium.

Composition and method for producing the same

The variant lipolytic enzymes provided herein may be used in the production of various compositions, such as enzyme compositions and cleaning or detergent compositions. Thus, in one embodiment, the present disclosure provides an enzyme composition comprising a variant lipolytic enzyme of the present disclosure, as well as cleaning or detergent compositions comprising a variant lipolytic enzyme provided herein or an enzyme composition comprising such a variant lipolytic enzyme.

As used herein, "enzyme composition" refers to any enzyme product, preparation, or composition comprising at least one of the variant lipolytic polypeptides provided herein. Such an enzyme composition may be a spent medium or filtrate containing one or more variant lipolytic enzymes, or one or more variant lipolytic enzymes and one or more additional enzymes. Spent medium means a medium comprising a host for the enzyme produced. Preferably, the host cells are separated from the culture medium after production. The enzyme composition may be a "whole culture broth" composition, optionally after inactivation of one or more production hosts or one or more microorganisms without any biomass separation, downstream processing, or purification of one or more desired variant lipolytic enzymes, as the variant polypeptides may be secreted into the culture medium and they exhibit activity under the environmental conditions of the used culture medium.

The enzyme composition may contain the variant lipolytic enzyme in at least partially purified and isolated form. It may even consist essentially of the desired enzyme or enzymes. The enzyme composition may be dried, spray dried or lyophilized, granulated, or the enzyme activity may be concentrated and/or stabilized in other ways for storage, if desired. If desired, the desired enzyme may be crystallized or isolated or purified according to conventional methods (e.g., filtration, extraction, precipitation, chromatography, affinity chromatography, electrophoresis, etc.).

The enzyme particles may be prepared, for example, by: rotary atomization, wet granulation, dry granulation, spray drying, disk granulation, extrusion, pan coating, spheronization, rotary drum granulation, fluid bed agglomeration, high shear granulation, fluid bed spray coating, crystallization, precipitation, emulsion gelation, rotary disk atomization, and other casting methods, and spheronization processes. The core of the particle may be the particle itself or the core of a layered particle.

In some embodiments, the enzyme composition comprises a variant lipolytic enzyme as provided herein in combination with one or more additional enzymes selected from the group consisting of: acyltransferases, alpha-amylases, beta-amylases, alpha-galactosidases, arabinosidases, aryl esterases, beta-galactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, endo-beta-1, 4-glucanases endo-beta-mannanase, esterase, exo-mannanase, feruloyl esterase, galactanase, glucoamylase, hemicellulase, enzyme preparation method, and pharmaceutical composition hexosaminidase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, mannanase metalloproteinases, nucleases (e.g., deoxyribonucleases and ribonucleases), oxidases, oxidoreductases, pectate lyases, pectoacetases, pectinases, pentosanases, perhydrolases, peroxidases, phenol oxidases, phosphatases, phospholipases, phytases, polygalacturonases, polyesterases, proteases, pullulanases, reductases, rhamnogalacturonases, beta-glucanases, tannase, transglutaminases, xylan acetyl esterases, xylanases, xyloglucanases, xylosidases, and any combination or mixture thereof. Typically, the at least one enzyme coating comprises at least one variant lipolytic enzyme.

The enzyme composition may be in any suitable form. For example, the enzyme composition may be in the form of a liquid composition or a solid composition, such as a solution, dispersion, paste, powder, granule, coated granule, tablet, cake, crystal slurry, gel, or pellet.

The enzyme composition may be used in a detergent or accelerator which is added outside the detergent during or before the washing process and which is for example in the form of a liquid, gel, powder, granule or tablet. The enzyme composition and detergent components may also be impregnated into a carrier such as a textile.

The present disclosure further provides cleaning or detergent compositions comprising a variant lipolytic enzyme as provided herein. The cleaning or detergent composition typically comprises a variant lipolytic enzyme as provided herein and one or more additional detergent components, such as a surfactant.

The present disclosure further includes a detergent or cleaning composition. As used herein, the term "detergent composition" or "detergent formulation" is used in reference to a composition intended for use in cleaning or treating a soiled or soiled object (including a particular textile or non-textile object or article) in a cleaning medium (e.g., a cleaning solution). Such compositions of the present invention are not limited to any particular detergent composition or formulation. Indeed, in some embodiments, the detergents of the present invention comprise at least one variant lipolytic enzyme as provided herein, additionally comprising one or more surfactants, one or more transferases, additional hydrolases, oxidoreductases, builders (e.g., builder salts), bleaching agents, bleach activators, bluing agents, fluorescent dyes, caking inhibitors, masking agents, enzyme activators, antioxidants and/or solubilizing agents. In some cases, the builder salt is a mixture of silicate and phosphate, preferably having more silicate (e.g., sodium metasilicate) than phosphate (e.g., sodium tripolyphosphate). Some compositions of the present invention, such as but not limited to cleaning compositions or detergent compositions, do not contain any phosphate (e.g., phosphate or phosphate builder).

Compositions having variant lipolytic enzymes which may be used in the methods provided herein may comprise use of variant lipolytic enzymes at a concentration of 0.001 to 10,000mg/L, or 0.001 to 2000mg/L, or 0.01 to 5000mg/L, or 0.01 to 2000mg/L, or 0.01 to 1300mg/L, or 0.1 to 5000mg/L, or 0.1 to 2000mg/L, or 0.1 to 1300mg/L, or 1 to 5000mg/L, or 1 to 1300mg/L, or 1 to 500mg/L, or 10 to 5000mg/L, or 10 to 1300mg/L, or 10 to 500 mg/L. In another embodiment, the composition may contain a variant lipolytic enzyme in an amount of 0.002 to 5000mg of protein, such as 0.005 to 1300mg of protein, or 0.01 to 5000mg of protein, or 0.01 to 1300mg of protein, or 0.1 to 5000mg of protein, or 1 to 1300mg of protein, preferably 0.1 to 1300mg of protein, more preferably 1 to 1300mg of protein, even more preferably 10 to 500mg of protein per liter of wash liquor, or at least 0.01ppm of active lipase.

In one embodiment, the composition comprises a variant lipolytic enzyme as provided herein and at least one additional detergent component, and optionally one or more additional enzymes.

In some embodiments, the cleaning or detergent compositions of the present invention further comprise adjunct materials including, but not limited to, surfactants, builders, bleaches, bleach activators, bleach catalysts, other enzymes, enzyme stabilization systems, chelants, optical brighteners, soil release polymers, dye transfer agents, dispersants, suds suppressors, dyes, perfumes, colorants, filler salts, hydrotropes, photoactivators, fluorescers, fabric conditioning agents, hydrolyzable surfactants, preservatives, antioxidants, anti-shrinkage agents, anti-wrinkle agents, bactericides, fungicides, color stippling agents, silver care agents, antitarnish and/or anti-corrosion agents, alkalinity sources, solubilizing agents, carriers, processing aids, pigments and pH control agents (see, e.g., U.S. Pat. nos. 6,610,642, 6,605,458, 5,705,464, 5,710,115, 5,698,504, 5,695,679, 5,686,014 and 5,646,101, all of which are incorporated herein by reference).

The detergent or cleaning compositions of the present disclosure are advantageously used in, for example, laundry applications, hard surface cleaning, dishwashing applications, and decorative applications (such as denture, tooth, hair, and skin cleaning). In addition, in some embodiments, the variant lipolytic enzymes of the invention are ideally suited for laundry applications. Furthermore, variations of the present disclosure may be used in particulate and liquid compositions.

The enzyme component weight is based on total active protein. All percentages and ratios are by weight unless otherwise indicated. All percentages and ratios are calculated based on the total composition unless otherwise indicated. In laundry detergent compositions, enzyme levels are expressed in ppm, which is equivalent to mg active protein per kg of detergent composition.

In some embodiments, the laundry detergent compositions described herein further comprise a surfactant. In some embodiments, the surfactant is selected from the group consisting of nonionic surfactants, amphoteric surfactants, semi-polar surfactants, anionic surfactants, cationic surfactants, zwitterionic surfactants, and combinations and mixtures thereof. In yet further embodiments, the surfactant is selected from the group consisting of anionic surfactants, cationic surfactants, zwitterionic surfactants, and combinations thereof. In some embodiments, the laundry detergent compositions described herein comprise from about 0.1% to about 60%, from about 1% to about 50%, or from about 5% to about 40%, by weight of the composition, of surfactant.

Exemplary surfactants include, but are not limited to, sodium dodecyl benzene sulfonate, C12-14 alkanol polyether-7, C12-15 alkanol polyether sodium sulfate, C14-15 alkanol polyether-4, sodium laureth sulfate (e.g., steol CS-370), sodium hydrogenated cocoate, C12 ethoxylate (Alfonic 1012-6, hetoxol LA7, hetoxol LA 4), sodium alkyl benzene sulfonate (e.g., nacconol 90G), and combinations and mixtures thereof. Anionic surfactants include, but are not limited to, linear Alkylbenzene Sulfonate (LAS), alpha-olefin sulfonate (AOS), alkyl sulfate (fatty Alcohol Sulfate) (AS), alcohol ethoxy sulfate (AEOS or AES), secondary Alkane Sulfonate (SAS), alpha-sulfo fatty acid methyl ester, alkyl-or alkenyl succinic acid, or soap. Nonionic surfactants include, but are not limited to, alcohol ethoxylates (AEO or AE), carboxylated alcohol ethoxylates, nonylphenol ethoxylates, alkyl polyglycosides, alkyl dimethylamine oxides, ethoxylated fatty acid monoethanolamides, polyhydroxy alkyl fatty acid amides (e.g., as described in WO 92/06154), polyoxyethylene esters of fatty acids, polyoxyethylene sorbitan esters (e.g., TWEEN), polyoxyethylene alcohols, polyoxyethylene iso-alcohols, polyoxyethylene ethers (e.g., TRITON and BRIJ), polyoxyethylene esters, polyoxyethylene-P-tert-octylphenol or octylphenyl-ethylene oxide condensates (e.g., nodet P40), condensates of ethylene oxide with fatty alcohols (e.g., LUBROL), polyoxyethylene nonylphenol, polyalkylene glycols (synmeronic F108), glycosyl surfactants (e.g., glucopyranoside, thiopyranoside), and combinations and mixtures thereof.

In further embodiments, the laundry detergent compositions described herein further comprise a mixture of surfactants including, but not limited to, 5% -15% anionic surfactant, <5% nonionic surfactant, cationic surfactant, phosphonate, soap, enzyme, perfume, butylphenyl methyl propionate, geraniol, zeolite, polycarboxylate, hexyl cinnamaldehyde, limonene, cationic surfactant, citronellol, and benzisothiazolinone.

The laundry detergent compositions described herein may additionally comprise one or more detergent builders or builder systems, complexing agents, polymers, bleach systems, stabilizers, suds boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, hydrotropes, optical brighteners, fabric conditioning agents and perfumes. As provided in more detail herein, the laundry detergent compositions described herein may further comprise an additional enzyme selected from the group consisting of a protease, an amylase, a cellulase, a lipase, a mannanase, a nuclease, a pectinase, a xyloglucanase, or a perhydrolase.

In some embodiments, the laundry detergent compositions described herein further comprise from about 1%, from about 3% to about 60%, or even from about 5% to about 40%, by weight of the cleaning composition, of a builder. Builders can include, but are not limited to, alkali metal, ammonium and alkanolammonium salts of polyphosphates; alkali metal silicates, alkaline earth metals and alkali metal carbonates; an aluminosilicate; a polycarboxylate compound; ether hydroxy polycarboxylic esters; copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1,3, 5-trihydroxybenzene-2, 4, 6-trisulfonic acid, and carboxymethyl oxy succinic acid; various alkali metal, ammonium and substituted ammonium salts of polyacetic acids such as ethylenediamine tetraacetic acid and nitrilotriacetic acid; and polycarboxylic esters such as mellitic acid, succinic acid, citric acid, oxo disuccinic acid (oxydisuccinic acid), polymaleic acid, benzene 1,3, 5-tricarboxylic acid, carboxymethyl oxysuccinic acid, and soluble salts thereof.

In some embodiments, the builder forms water-soluble hardness ion complexes (e.g., chelating builders), such as citrates and polyphosphates (e.g., sodium tripolyphosphate and sodium tripolyphosphate hexahydrate, potassium tripolyphosphate, and mixed sodium tripolyphosphate and potassium tripolyphosphate, etc.). Any suitable builder may be used in the compositions described herein, including those known in the art.

In some embodiments, the laundry detergent compositions described herein further comprise adjunct ingredients including, but not limited to, surfactants, builders, bleaching agents, bleach activators, bleach catalysts, additional enzymes, enzyme stabilizers (including, for example, enzyme stabilizing systems), chelating agents, optical brighteners, soil release polymers, dye transfer agents, dye transfer inhibitors, catalytic materials, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay soil removal agents, structure elasticizing agents, dispersing agents, suds suppressors, dyes, perfumes, colorants, filler salts, hydrotropes, photoactivators, fluorescers, fabric conditioners, hydrolyzable surfactants, solvents, preservatives, antioxidants, anti-shrinkage agents, anti-wrinkle agents, bactericides, fungicides, color spotters, anti-corrosion agents, alkalinity sources, solubilizing agents, carriers, processing aids, pigments, pH control agents, and combinations thereof. (see, e.g., US6610642, US6605458, US5705464, US5710115, US5698504, US5695679, US5686014, and US 5646101). In some embodiments, one or more adjuvants are incorporated, for example, to aid or enhance cleaning performance (for treating the substrate to be cleaned), or to improve the aesthetics of the cleaning composition (e.g., as is the case with perfumes, colorants, dyes, etc.). Any such adjunct ingredient is in addition to the variant enzymes provided herein. In some embodiments, the adjunct ingredient is selected from the group consisting of surfactants, enzyme stabilizers, builder compounds, polymer compounds, bleaching agents, additional enzymes, suds suppressors, dispersants, lime-soap dispersants, soil suspending agents, softeners, anti-redeposition agents, corrosion inhibitors, and combinations thereof.

In some further embodiments, the laundry detergent compositions described herein comprise one or more enzyme stabilizers. In some embodiments, the enzyme stabilizer is a water-soluble source of calcium and/or magnesium ions. In some embodiments, these enzyme stabilizers include oligosaccharides, polysaccharides, and inorganic divalent metal salts (including alkaline earth metal salts, such as calcium salts). In some embodiments, enzymes used herein are stabilized by the presence of water-soluble sources of zinc (II), calcium (II), and/or magnesium (II) ions, as well as other metal ions (e.g., barium (II), scandium (II), iron (II), manganese (II), aluminum (III), tin (II), cobalt (II), copper (II), nickel (II), and vanadyl (IV)) in finished compositions that provide such enzymes with such ions. Chlorides and sulphates may also be used in some embodiments. Exemplary oligosaccharides and polysaccharides (e.g., dextrins) are described, for example, in WO 07145964. In some embodiments, the laundry detergent compositions described herein contain reversible protease inhibitors selected from boron-containing compounds (e.g., borates, 4-formylphenylboronic acids, and phenylboronic acid derivatives, such as described in WO9641859, for example), peptide aldehydes (such as described in WO 2009118375 and WO 2013004636, for example), and combinations thereof.

The cleaning compositions herein are typically formulated such that the pH of the wash water is from about 3.0 to about 11 during use in an aqueous cleaning operation. The liquid product formulation is typically formulated to have a net pH of from about 5.0 to about 9.0, more preferably from about 7.5 to about 9. Particulate laundry products are typically formulated to have a pH of from about 8.0 to about 11.0. Techniques for controlling the pH at recommended use levels include the use of buffers, bases, acids, and the like, and are well known to those skilled in the art.

Suitable high pH cleaning compositions typically have a net pH of from about 9.0 to about 11.0, or even a net pH of from 9.5 to 10.5. Such cleaning compositions typically comprise a sufficient amount of a pH adjuster (such as sodium hydroxide, monoethanolamine, or hydrochloric acid) to provide such cleaning compositions with a net pH of from about 9.0 to about 11.0. Such compositions typically comprise at least one alkali stable enzyme. In some embodiments, the composition is a liquid, while in other embodiments, the composition is a solid.

In one embodiment, the cleaning compositions include those having a pH of from 7.4 to 11.5, or 7.4 to 11.0, or 7.5 to 11.5, or 7.5 to 11.0, or 7.5 to 10.5, or 7.5 to 10.0, or 7.5 to 9.5, or 7.5 to 9.0, or 7.5 to 8.5, or 7.5 to 8.0, or 7.6 to 11.5, or 7.6 to 11.0, or 7.6 to 10.5, or 8.7 to 10.0, or 8.0 to 11.5, or 8.0 to 11.0, or 8.0 to 10.0, or 8.5 to 10.5, or 8.0 to 10.0.

The concentration of the detergent composition in a typical wash solution throughout the world varies from less than about 800ppm of the detergent composition ("low detergent concentration geographical location") (e.g., about 667ppm in japan) to between about 800ppm and about 2000ppm ("medium detergent concentration geographical location") (e.g., about 975ppm in the united states, about 1500ppm in brazil), to greater than about 2000ppm ("high detergent concentration geographical location") (e.g., about 4500ppm to about 5000ppm in europe, about 6000ppm in high foam phosphate builder geographical location).

In some embodiments, the detergent compositions described herein may be used at temperatures ranging from about 10 ℃ to about 60 ℃, or from about 20 ℃ to about 60 ℃, or from about 30 ℃ to about 60 ℃, from about 40 ℃ to about 55 ℃, or at all ranges within 10 ℃ to 60 ℃. In some embodiments, the detergent compositions described herein are used in a "cold water wash" at temperatures ranging from about 10 ℃ to about 40 ℃, or from about 20 ℃ to about 30 ℃, from about 15 ℃ to about 25 ℃, from about 15 ℃ to about 35 ℃, or at all ranges within 10 ℃ to 40 ℃.

As a further example, different geographic locations typically have different water hardness. Ca generally mixed per gallon ²⁺ /Mg ²⁺ The number of particles to describe the water hardness. Hardness is calcium (Ca) in water ²⁺ ) And magnesium (Mg) ²⁺ ) Is a measure of the amount of (a). In the united states, most water is hard water, but the hardness varies. Medium hard (60-120 ppm) to hard (121-181 ppm) water has hardness minerals of 60 to 181 parts per million (parts per million converted to particles per U.S. gallon is the number of ppm divided by 17.1 equals particles per gallon).

Table i water hardness level

Water and its preparation method	Particle/gallon	Parts per million
			Soft and soft	Less than 1.0	Less than 17
Slightly harder	1.0 to 3.5	17 to 60
			Medium hard	3.5 to 7.0	60 to 120
Hard	7.0 to 10.5	120 to 180
			Very hard	Greater than 10.5	Greater than 180

Typically, european water hardness is greater than about 10.5 (e.g., about 10.5 to about 20.0) particles/gallon of mixed Ca ²⁺ /Mg ²⁺ (e.g., about 15 particles/gallon mixed Ca) ²⁺ /Mg ²⁺ ). Typically, north american water hardness is greater than japanese water hardness but less than european water hardness. For example, the north american water hardness may be between about 3 to about 10 particles, about 3 to about 8 particles, or about 6 particles. Typically, japanese water hardness is lower than North America water hardness, typically less than about 4, e.g., about 3 particles/gallon mixed Ca ²⁺ /Mg ²⁺ 。

In other embodiments, the compositions described herein comprise one or more additional enzymes. The one or more additional enzymes are selected from the group consisting of acylases, alpha-amylases, beta-amylases, alpha-galactosidases, arabinosidases, aryl esterases, beta-galactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, dnases, endo-beta-1, 4-glucanases, endo-beta-mannanases, esterases, exo-mannanases, galactanases, glucoamylases, hemicellulases, hexosaminidases, hyaluronidases, keratinases, laccases, lactases, ligninases, lipases, lipoxygenases, mannanases, metalloproteinases, nucleases (e.g., deoxyribonucleases and ribonucleases), oxidases, oxidoreductases, pectate lyases, pectinacetylesterases, pectinases, pentosanases, peroxidases, phenol oxidases, phosphatases, phytases, polygalacturonases, polysaccharidases, additional proteases, pullulanases, reductases, rhamnomannanases, mannanases, xylanases, and mixtures thereof. Some embodiments relate to a combination (i.e., a "mixture") of enzymes (such as amylase, protease, lipase, mannanase and/or nuclease) that bind to one or more variant lipolytic enzymes in the compositions provided herein.

In some embodiments, the compositions provided herein comprise a variant lipolytic enzyme in combination with a protease. Proteases for use in combination with the variant lipolytic enzyme in the compositions of the present disclosure include any polypeptide having protease activity. In one embodiment, the additional protease is a serine protease. In another embodiment, the additional protease is a metalloprotease, a fungal subtilisin, or an alkaline microbial protease or a trypsin-like protease. Suitable proteases include those of animal, plant or microbial origin. In some embodiments, the protease is a microbial protease. In other embodiments, the protease is a chemically or genetically modified mutant. In another embodiment, the protease is a subtilisin-like protease or a trypsin-like proteinAn enzyme. In other embodiments, the additional protease does not contain epitopes that cross-react with the variant, as measured by antibody binding or other assays available in the art. Exemplary subtilisins include those derived from, for example, bacillus (e.g., BPN', jiamber (Carlsberg), subtilisin 309, subtilisin 147, and subtilisin 168) or fungal sources, such as, for example, those described in U.S. patent No. 8,362,222. Exemplary additional proteases include, but are not limited to, WO92/21760, WO95/23221, WO2008/010925, WO 09/1494200, WO 09/14949, WO 09/1494145, WO 10/056640, WO10/056653, WO2010/0566356, WO11/072099, WO2011/13022, WO11/140364, WO 12/151534, WO2015/038792, WO2015/089447, WO2015/089441, WO2017/215925, U.S. publication No. 2008/0090747, U.S. 5,801,039, U.S. 5,340,735, U.S. Pat. No. 5,500,364, U.S. Pat. No. 5,855,625, RE 34,606, U.S. Pat. No. 5,955,340, U.S. Pat. No. 5,700,676; U.S. Pat. No. 6,312,936, U.S. Pat. No. 6,482,628, U.S. Pat. No. 8,530,219, U.S. provisional application Nos. 62/180673 and 62/161077, and those described in PCT application Nos. PCT/US2015/021813, PCT/US2015/055900, PCT/US2015/057497, PCT/US2015/057512, PCT/US2015/057526, PCT/US2015/057520, PCT/US2015/057502, PCT/US2016/022282 and PCT/US16/32514, international publications WO 2016001449, WO 2016087617, WO 2016096714, WO 2016203064, WO 2017089093, and WO 2019180111, and metalloproteases described in WO 1999014341, WO 1999033960, WO 1999014342, WO 1999034003, WO 2007044993, WO 2009058303, WO 2009058661, WO 2014071410, WO 2014194032, WO 2014194034, WO 2014194054 and WO 2014/194117. Exemplary additional proteases include, but are not limited to, trypsin (e.g., of porcine or bovine origin) and Fusarium (Fusarium) protease described in WO 89/06270. Exemplary commercial proteases include, but are not limited to MAXACAL ^TM 、MAXAPEM ^TM 、 OXP、PURAMAX ^TM 、EXCELLASE ^TM 、PREFERENZ ^TM Proteases (e.g., P100, P110, P280), EFFECTENZ ^TM Proteases (e.g. P1000, P1050, P2000), EXCELLENZ ^TM Proteases (e.g.P1000), -or-a->And PURAFAST ^TM (DuPont));Variants(s),16L、 ULTRA、 DURAZYM ^TM 、

PROGRESSAnd->(Novozymes corporation); BLAP (blast furnace potential) ^TM And BLAP ^TM Variants (Henkel); LAVERGY ^TM PRO 104L、LAVERGY ^TM PRO 106LS、LAVERGY ^TM PRO114LS (BASF), KAP (Bacillus alcaligenes subtilisin (Kao) and +.>(AB enzyme preparation Co., ltd.).

In some embodiments, the compositions provided herein comprise a variant lipolytic enzyme in combination with one or more amylases. In one embodiment, the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% amylase by weight of the composition. Any amylase (e.g., an alpha amylase and/or a beta amylase) suitable for use in alkaline solutions may be used for inclusion in such compositions. Exemplary amylases may be chemically or genetically modified mutants. Exemplary amylases include, but are not limited to, those of bacterial or fungal origin, such as, for example, the amylases described in: GB 1,296,839, WO9402597, WO WO, WO-based, WO, WO WO, WO WO, WO-based, WO WO, WO-based, WO, WO WO, WO 419. WO 2010/059413, WO 2010088447, WO 2010091221, WO 2010104675, WO 2010115021, WO10115028, WO 2010117511, WO 2011076123, WO 2011076897, WO 2011080352, WO 2011080353, WO 2011080354, WO 2011082425, WO 2011082429, WO 2011087836, WO 2011098531, WO 2013063460, WO 2013184577, WO 2014099523, WO 2014164777, and WO 2015077126. Exemplary commercial amylases include, but are not limited to STAINZYMESTAINZYMESTAINZYMEAnd BAN ^TM (Norwechat corporation); EFFECTENZ ^TM S1000、POWERASE ^TM 、PREFERENZ ^TM S100、PREFERENZ ^TM S110、EXCELLENZ ^TM S2000、And->P (DuPont).

In some embodiments, the compositions provided herein comprise a variant lipolytic enzyme in combination with one or more additional lipases. In some embodiments, the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% lipase by weight of the composition. Exemplary lipases may be chemically or genetically modified mutants. Exemplary lipases include, but are not limited to, for example, bacteria or fungiThose of origin, for example, such as Humicola lanuginosa (H.lanuginosa) lipases (see, for example, EP 258068 and EP 305116), thermomyces lanuginosus (T.lanuginosa) lipases (see, for example, WO 2014/059360 and WO 2015/010009), rhizomucor miehei (Rhizomucor miehei) lipases (see, for example, EP 238023), candida (Candida) lipases such as Candida antarctica (C.antarctica) lipases (such as Candida antarctica lipase A or B) (see, for example, EP 214761), pseudomonas lipases such as Pseudomonas alcaligenes (P.alcaligenes) and Pseudomonas alcaligenes) (see, for example, EP 218272), pseudomonas cepacia (P.cepacia) lipases (see, for example, EP 331376), pseudomonas (P.stutzeri) lipases (such as Candida antarctica (C.antarctica) lipases (such as Candida antarctica lipase A or B) (see, for example, EP 214761, pseudomonas pseudoalcaligenes) and Pseudomonas pseudoalcaligenes (P.pseudoalcaligenes) lipases (see, for example, P.218272), pseudomonas cepacia (see, P.P.cepacia) lipases (see, for example, bacillus sp. Biol) and Bacillus sp. Biol (Biopsilosis) lipase (see, 3, bacillus sp. Biol. Lipase, B, and so on ]1131:253-260 (1993)), bacillus stearothermophilus lipase (see, e.g., JP 64/744992), and Bacillus pumilus (B.pumilus) lipase (see, e.g., WO 91/16422). Exemplary cloned lipases include, but are not limited to, penicillium sambac (Penicillium camembertii) lipase (see Yamaguchi et al, gene [ Gene ]]103:61-67 (1991)); geotrichum candidum (Geotrichum candidum) lipase (see Schimada et al, J.biochem. [ J.Biochem.)]383-388 (1989)); and various Rhizopus (Rhizopus) lipases, such as Rhizopus delbrueckii (R. Delete) lipases (see Hass et al, gene [ Gene ]]109:117-113 (1991)), rhizopus niveus (R.niveus) lipase (Kugimiya et al, biosci. Biotech Biochem. [ bioscience, biotechnology and biochemistry ]]56:716-719 (1992)) and Rhizopus oryzae (R.oryzae) lipase. Other lipolytic enzymes (e.g., cutinases) may also be used in one or more of the compositions described herein, including but not limited to cutinases derived from Pseudomonas mendocina (Pseudomonas mendocina) (see WO 88/09367) and/or Fusarium pisiformis (Fusarium solani pisi) (see WO 90/09446), for example. Exemplary commercial LIPASEs include, but are not limited to, M1 LIPASE ^TM 、LUMA FAST ^TM And LIPOMAX ^TM (DuPont company);andULTRA (Norwechat Co.); LIPASE P ^TM (Tianye pharmaceutical Co., ltd. (Amano Pharmaceutical Co. Ltd)).

In some embodiments, the compositions provided herein comprise a variant lipolytic enzyme in combination with one or more mannanases. In one embodiment, the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% mannanase enzyme by weight of the composition. Exemplary mannanases may be chemically or genetically modified mutants. Exemplary mannanases include, but are not limited to, those of bacterial or fungal origin, such as, for example, those described in the following: WO 2016/007929; USPN 6,566,114;6,602,842; and 6,440,991; U.S. provisional application nos. 62/251516, 62/278383, and 62/278387. Exemplary commercial mannanases include, but are not limited to(Norwechat corporation) and EFFECTENZ ^TM M 1000、EFFECTENZ ^TM M 2000、M 100、And PURABRITE ^TM (DuPont company). />

In some embodiments, the compositions and methods provided herein comprise a variant lipolytic enzyme in combination with a nuclease (e.g., dnase or rnase). Exemplary nucleases include, but are not limited to, those described in WO 2015181287, WO 2015155350, WO 2016162556, WO 2017162836, WO 2017060475 (e.g., SEQ ID NO: 21), WO 2018184816, WO 2018177936, WO 2018177938, WO2018/185269, WO 2018185285, WO 2018177203, WO 2018184817, WO 2019084349, WO 2019084350, WO 2019081721, WO 2018076800, WO 2018185267, WO 2018185280, and WO 2018206553. Other nucleases that can be used in combination with the variant lipolytic enzymes in the compositions and methods provided herein include those described in the following: nijland R, hall MJ, burgess JG (2010) Dispersal of Biofilms by Secreted, matrix Degrading, bacterial DNase [ biofilm by secretion, matrix degradation, bacterial DNase dispersion ]. PLoS ONE [ public science library: synthesis ]5 (12) and Whithurch, C.B., tolker-Nielsen, T., ragas, P.C., mattick, J.S. (2002) Extracellular DNA required for bacterial biofilm formation [ extracellular DNA required for bacterial biofilm formation ]. Science [ Science ]295:1487.

Still further embodiments relate to compositions comprising one or more variant lipolytic enzymes described herein and one or more cellulases. In one embodiment, the composition comprises from about 0.00001% to about 10%, 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% cellulase by weight of the composition. Any suitable cellulase may be used in the compositions described herein. Exemplary cellulases may be chemically or genetically modified mutants. Exemplary cellulases include, but are not limited to, those of bacterial or fungal origin, such as, for example, those described in the following: WO 2005054475, WO 2005056787, US 7,449,318, US 7,833,773, US 4,435,307; EP 0495257; and U.S. provisional application No. 62/296,678. Exemplary commercial cellulases include, but are not limited to And->PREMUM (Norvigilance Co.); REVITALENZ ^TM 100、REVITALENZ ^TM 200/220, and->2000 (DuPont company); and KAC-500 (B) ^TM (Kao Corporation). In some embodiments, the cellulase is incorporated as part or fragment of a mature wild-type or variant cellulase in which a portion of the N-terminus is deleted (see, e.g., US 5,874,276).

In some embodiments, the laundry detergent compositions described herein comprise at least one chelant. Suitable chelating agents can include, but are not limited to, copper, iron, and/or manganese chelating agents, and mixtures thereof. In some embodiments, the laundry detergent compositions described herein comprise from about 0.1% to about 15%, or even from about 3.0% to about 10%, by weight of the composition, of the chelant.

In some still further embodiments, the laundry detergent compositions described herein comprise at least one deposition aid. Suitable deposition aids include, but are not limited to, polyethylene glycol, polypropylene glycol, polycarboxylates, soil release polymers (such as polyethylene terephthalate), clays (such as kaolin), montmorillonite, attapulgite, illite, bentonite, halloysite, and mixtures thereof.

In some embodiments, the laundry detergent compositions described herein comprise at least one anti-redeposition agent.

In some embodiments, the laundry detergent compositions described herein comprise one or more dye transfer inhibitors. Suitable polymeric dye transfer inhibitors include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones, and polyvinylimidazoles, or mixtures thereof. In some embodiments, the laundry detergent compositions described herein comprise from about 0.0001% to about 10%, from about 0.01% to about 5%, or even from about 0.1% to about 3%, by weight of the composition, of dye transfer inhibiting agent.

In some embodiments, the laundry detergent compositions described herein comprise one or more silicates. In some such embodiments, sodium silicate (e.g., sodium disilicate, sodium metasilicate, and crystalline layered silicate) may be used. In some embodiments, the laundry detergent compositions described herein comprise from about 1% to about 20%, or from about 5% to about 15%, by weight of the composition, of silicate salt.

In yet further embodiments, the laundry detergent compositions described herein comprise one or more dispersants. Suitable water-soluble organic materials include, but are not limited to, homo-or co-polymeric acids or salts thereof, wherein the polyacid comprises at least two carboxyl groups separated from each other by no more than two carbon atoms.

In some embodiments, the laundry detergent compositions described herein comprise one or more bleaching agents, bleach activators, and/or bleach catalysts. In some embodiments, the laundry detergent compositions described herein comprise one or more inorganic and/or organic bleaching compounds. Inorganic bleaching agents may include, but are not limited to, perhydrate salts (e.g., perborates, percarbonates, perphosphates, persulfates, and persilicates). In some embodiments, the inorganic perhydrate salt is an alkali metal salt. In some embodiments, the inorganic perhydrate salt is included as a crystalline solid without additional protection, but in some other embodiments, the salt is coated. Suitable salts include, for example, those described in EP 2100949. Bleach activators are typically organic peracid precursors that enhance bleaching during cleaning at temperatures of 60 ℃ and below. Bleach activators suitable for use herein include compounds which under perhydrolysis conditions give aliphatic peroxycarboxylic acids and/or optionally substituted peroxybenzoic acids preferably having from about 1 to about 10 carbon atoms, especially from about 2 to about 4 carbon atoms. Bleach catalysts typically include, for example, manganese triazacyclononane and related complexes, and cobalt, copper, manganese and iron complexes, as well as those described in US4246612, US5227084, US4810410, WO9906521 and EP 2100949.

In some embodiments, the laundry detergent compositions described herein comprise one or more catalytic metal complexes. In some embodiments, a metal-containing bleach catalyst may be used. In other embodiments, the metal bleach catalyst comprises a catalytic system comprising: transition metal cations with defined bleach catalytic activity (e.g. copper, iron, titanium, ruthenium, tungsten, molybdenum or manganese cations), auxiliary metal cations with little or no bleach catalytic activity (e.g. zinc or aluminium cations), and chelates with defined stability constants for the catalytic and auxiliary metal cations, in particular ethylenediamine tetraacetic acid, ethylenediamine tetra (methylenephosphonic acid) and water-soluble salts thereof (see e.g. US 4430243). In some embodiments, the laundry detergent compositions described herein are catalyzed by a manganese compound. Such compounds and use levels are well known in the art (see, e.g., US 5576282). In further embodiments, cobalt bleach catalysts may be used in the laundry detergent compositions described herein. Various cobalt bleach catalysts are known in the art (see, e.g., US5597936 and US 5595967) and are readily prepared by known procedures.

Polyesters as used herein include polymers containing at least one ester repeat unit in their backbone polymer. In its simplest form, the polyester is produced by polycondensation of ethylene glycol (diol) with a dicarboxylic acid (diacid) or diester thereof. Polyesters include naturally occurring chemicals, such as those found in the cutin of the plant cuticle, and synthetic materials such as polybutyrates by step-growth polymerization (step-growth polymerization).

Polyesters that may be contacted with the variant lipases provided herein (e.g., in the methods provided herein), or compositions comprising such variant lipases, include any ester bond-containing polymer. Such polyesters include aliphatic and aromatic polyesters. The aliphatic polyester includes: polyhydroxyalkanoates (PHA), which can be classified into Polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH), and copolymers thereof; polylactic acid (PLA); poly (epsilon-caprolactone) (PCL); polybutylene succinate (PBS) and its derivatives poly (butylene succinate adipate) (PBSA). The aromatic polyester includes: modified poly (ethylene terephthalate) (PET), such as poly (butylene adipate terephthalate) (PBAT) and poly (tetramethylene adipate-co-terephthalate) (PTMAT); and aliphatic-aromatic copolyesters (AAC). In some embodiments, the polyester may be partially or substantially biodegradable. In other embodiments, the polyester may be partially or substantially resistant to microbial and enzymatic attack.

In some embodiments, the polyester may be an aliphatic polyester. In some embodiments, the polyester may be an aromatic polyester. In some embodiments, the aromatic polyester may be polyethylene terephthalate (PET). In some embodiments, the aromatic polyester may be polytrimethylene terephthalate (PTT).

Thus, in one embodiment, polyesters useful in the methods provided herein include those selected from the group consisting of: polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), polyethylene naphthalate (PEN), polyester polyurethane, poly (ethylene adipate) (PEA), and combinations thereof.

In another embodiment, the fabrics or textiles useful in the methods provided herein include fabrics and textiles comprising at least one polyester selected from the group consisting of: polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), polyethylene naphthalate (PEN), polyester polyurethane, poly (ethylene adipate) (PEA), and combinations thereof.

In some embodiments, the present disclosure provides methods for treating a fabric or textile comprising contacting the fabric or textile with a variant lipolytic enzyme as provided herein, or a composition comprising such a variant lipolytic enzyme, and optionally rinsing the fabric or textile.

In some embodiments, the contacting step of the methods provided herein comprises a variant lipolytic enzyme in an amount selected from the group consisting of: 0.002 to 10,000mg of protein, 0.005 to 5000mg of protein, 0.01 to 5000mg of protein, 0.05 to 1300mg of protein, 0.1 to 500mg of protein, 0.1 to 100mg of protein per liter of wash solution.

Esterases for surface modification

In some embodiments, a polyester (e.g., PET) containing textile, fabric, or film may have a hydrolyzable polymer end or ring on its surface. The variant lipolytic enzymes provided herein are useful in methods of surface modification of polyester (e.g., PET) fibers, which may improve factors such as after-finishing fastness (finishing fastness), dyeability, wettability, depilling, and anti-pilling. In some embodiments, polymer chains protruding or forming loops on the surface of textiles, fibers, or films comprising polyesters (e.g., PET) can be hydrolyzed by the variant lipases herein to carboxylic acid and hydroxyl residues, thereby increasing surface hydrophilicity. Pilling is the formation of small pills on the surface of polyester (e.g., PET) fabrics, resulting in an unsightly fraying appearance of the fabric. Typically, these spheres are produced from loose fibers in the fabric or fibers released from the tissue.

Thus, in some embodiments, the variant lipolytic enzymes of the present disclosure may be used in methods for post-finishing fastness, dyeability, wettability, depilling and anti-pilling of polyester (e.g., PET) textiles, fabrics, and films. In other embodiments, the variant lipolytic enzymes of the present disclosure may be used in detergent compositions to reduce pilling during textile cleaning. In some embodiments, the variant lipolytic enzyme of the present disclosure has PET enzymatic activity.

In one embodiment, methods for degrading polyesters or polyester-containing materials are provided, wherein the methods comprise contacting the polyester-containing material with a variant lipolytic enzyme or a composition comprising a variant lipolytic enzyme as provided herein. In some embodiments, the polyester-containing material is a polyester textile or fabric.

In another embodiment, the present disclosure provides a method for enzymatic depolymerization of a polyester-containing material, wherein the method comprises contacting the polyester-containing material with a variant lipolytic enzyme or a composition comprising a variant lipolytic enzyme as provided herein, and recovering monomers and/or oligomers of the polyester. In some embodiments, the polyester-containing material is a polyester textile or fabric.

In other embodiments, the variant lipolytic enzymes of the present disclosure may be used in methods for cleaning or conditioning textiles or fabrics, improving the thermo-physiological properties (e.g., heat or humidity management or wearing comfort) of textiles or fabrics comprising polyesters, and increasing the hydrophilicity of textiles or fabrics comprising polyesters. In other embodiments, the variant lipolytic enzymes of the present disclosure may be used in detergent compositions to clean or condition textiles or fabrics, to improve the thermo-physiological properties (e.g., heat or humidity management or wear comfort) of textiles or fabrics comprising polyesters, and to increase the hydrophilicity of textiles or fabrics comprising polyesters. In some embodiments, the variant lipolytic enzyme of the present disclosure has PET enzymatic activity.

In other embodiments, the variant lipolytic enzymes of the present disclosure may be used in methods for reducing the pilling effect and/or increasing the anti-ashing effect of a cleaning composition on a textile or fabric comprising polyester. In other embodiments, the variant lipolytic enzymes of the present disclosure may be used in detergent compositions to reduce pilling and/or increase anti-ashing effects of the detergent compositions on textiles or fabrics comprising polyesters. In some embodiments, the variant lipolytic enzyme of the present disclosure has PET enzymatic activity. In some embodiments, a variant lipolytic enzyme of the present disclosure is combined with a second enzyme (e.g. a cellulase).

The textile or fabric may be contacted with the variant lipolytic enzyme or a composition comprising the variant lipolytic enzyme in a washing machine or a manual washing tub (e.g. for hand washing). In one embodiment, the textile or fabric is contacted with a variant lipolytic enzyme or a composition comprising the variant lipolytic enzyme in a washing liquid. In another embodiment, the solution containing the variant lipolytic enzyme is incubated with or flowed through the polyester-containing material, such as by pumping the solution through a tube or pipe or by filling a reservoir with the solution.

In some embodiments, the textile or article is contacted with the variant lipolytic enzyme or the composition comprising the variant lipolytic enzyme under temperature conditions which allow the variant lipolytic enzyme to be active. In some embodiments, the temperatures in the methods disclosed herein include those between 10 ℃ and 60 ℃, between 10 ℃ and about 45 ℃, between 15 ℃ and about 55 ℃, between 15 ℃ and about 50 ℃, between 15 ℃ and about 45 ℃, between 20 ℃ and about 60 ℃, between 20 ℃ and about 50 ℃, and between 20 ℃ and about 45 ℃.

The polypeptides, compositions and methods provided herein are useful in a wide range of applications requiring degradation of polyesters (e.g., PET), including household cleaning in washing machines, dishwashers and on household surfaces, for example.

Other aspects and embodiments of the compositions and methods of the present invention will be apparent from the foregoing description and the examples that follow. Various alternative embodiments beyond those described herein may be employed in practicing the present invention without departing from the spirit and scope of the invention. The claims, therefore, rather than the specific embodiments described herein, define the scope of the invention and, as such, methods and structures within the scope of the claims and their equivalents are covered thereby.

Examples

Example 1. A variant lipolytic enzyme comprising an amino acid sequence having at least 70% identity to the full length amino acid sequence of SEQ ID NO. 2, the variant lipolytic enzyme comprising the substitutions T064V-T117L-T177N/R-I178L-F180P-Y182A-R190L-S205G-S212D-F226L-Y239I-L249P-S252I-L258F and further comprising at least one additional substitution selected from the group consisting of: V014S, R040A/T, G059Y, G061D, A066D, S070E, Q161H, G A/E, F207TL/T, V210I, Q227H, A236P, S244E, E Q and R256K, wherein said positions are numbered by reference to the amino acid sequence of SEQ ID NO:2 and wherein said variant has esterase activity.

Example 2. The variant lipolytic enzyme of example 1, wherein the variant comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the full-length amino acid sequence of SEQ ID NO. 2.

Example 3. The variant lipolytic enzyme of example 1 or 2, wherein the variant is derived from a parent enzyme comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the full-length amino acid sequence of SEQ ID NO. 2.

Embodiment 4 the variant lipolytic enzyme of any of the preceding embodiments, wherein the variant comprises a combination of substitutions selected from the group consisting of: R40T-T64V-T117T-G175L-T180G-G175A-R190G-S205G-F207L-S212D-F226L-Y239I-L249P-S252I-L258F, R T-G61D-T64V-S70E-T117L-T177L-S178L-F180P-Y180A-R190L-S205G-F207T-S212D-F226L-Q227H-A236P-Y239I-L249P-S252I-E258Q-L F, R T64T-T64V-S70E-T117L-T117N-I178L-F182A-R182L-S205T-F207T-S212D-F226L-A236P-Y249P-S252I-E254Q-L5740A-T64V-E70T-T180F 180L-T180I-S182A-L182-D180F-L180; F226L-A236P-Y239I-L249P-S252I-E254Q-L258F, R T-T64V-S70E-T117L-Q161H-T177N-I178L-F180P-Y182A-R190L-S205G-F207T-S212D-F226L-A236P-Y239I-L249P-S252I-E254Q-L258F, R T-T64V-S70E-T117L-G117A-T177N-I178L 180P-Y182A-R190L-S205G-F207T-S212D-F226L-A236P-Y239I-L249P-S252P-E254Q-L258F, R T64V-S70E-T117L-T177N-I178L 180P-Y182A-R190L-S190G-S205T 210F 180P-E252A-F252D 252L-E258P-E252L-E252L-I254, R40T-T64V-T70E-T117L-T178L-F180P-Y182A-R190L-S205G-F207T-S212D-F226L-A236P-Y239I-S244E-L249P-S254I-E254Q-L258T 40T-T64V-S70E-T117L-T177N-I178L-F180P-Y182A-R190L-S205G-F207T-S212D-F226L-A236P-Y249P-S254Q-R256K-L258F, V A-G61Y-G61D-T64V-A66D-S70E-T117L-Q161H-T177R-I178L-F180P-Y180A-R190L-S205G-F207T-S210I-S212D-F226L-A236P-Y239I-L249P-S252I-E254Q-R256K-E254K-T64V-S70E-T117S-R40A-G59Y-G61D-T64V-S70E-R190L-S205G-F207T-V210I-S212D-F226L-A236P-Y239I-L249P-S252I-E254Q-R256K-L258 6240T-G61D-T64V-S70E-T117L-Q161H-T177R-178L-F180P-Y182A-R205G-F205T-V210I-S212D-F226L-A236P-Y239I-L249P-S252I-E254Q-R256K-L258F 258, and V14S-R40A-G59Y-G61D-T64V-A66D-S70E-T117L-Q161H-G175A-T177R-I178L-F180P-Y182A-R190L-S205G-F207T-V210I-S212D-F226L-A236P-Y239I-L249P-S252I-E254Q-R256K-L258F, wherein the positions are numbered by reference to the amino acid sequence of SEQ ID NO: 2.

Embodiment 5 the variant lipolytic enzyme of any of the preceding embodiments, wherein the variant has one or more improved properties when compared to a parent or reference lipolytic enzyme, wherein the improved properties are selected from improved stability, improved hydrolytic activity towards polyesters, or a combination thereof.

Embodiment 6 the variant lipolytic enzyme of any of the preceding embodiments, wherein the improved property is:

(i) Improved stability, wherein the variant has a residual activity of at least 5% when measured according to the stability assay of example 3, and/or

(ii) Improved hydrolytic activity towards polyesters, wherein the variant has a PI.gtoreq.1.2 compared to a lipolytic enzyme having the amino acid sequence of SEQ ID NO:2 substituting R40T-T64V-T117L-T177N-I178L-F180P-Y182A-R190L-S205G-F207T-S212D-F226L-Y239I-L249P-S252I-L258F when measured according to the PET assay of example 2.

Embodiment 7. The lipolytic enzyme of any of embodiments 1-6, wherein the variant has hydrolytic activity on a polyester selected from the group consisting of: polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), polyethylene naphthalate (PEN), polyester polyurethane, poly (ethylene adipate) (PEA), and combinations thereof.

Embodiment 8. A polynucleotide comprising a nucleic acid sequence encoding the variant lipolytic enzyme according to any of embodiments 1-7.

Embodiment 9. The polynucleotide of embodiment 8 wherein the nucleic acid sequence is operably linked to a promoter.

Example 10. An expression vector or cassette comprising the polynucleotide of example 8 or 9.

Example 11 a recombinant host cell comprising an expression vector or cassette as described in example 10.

Embodiment 12. An enzyme composition comprising the variant lipolytic enzyme according to any of embodiments 1-7.

Embodiment 13. The enzyme composition of embodiment 12, wherein the composition further comprises at least one additional enzyme selected from the group consisting of: acyltransferases, alpha-amylases, beta-amylases, alpha-galactosidases, arabinosidases, aryl esterases, beta-galactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, endo-beta-1, 4-glucanases endo-beta-mannanase, esterase, exo-mannanase, feruloyl esterase, galactanase, glucoamylase, hemicellulase, enzyme preparation method, and pharmaceutical composition hexosaminidase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, mannanase metalloproteinases, nucleases (e.g., deoxyribonucleases and ribonucleases), oxidases, oxidoreductases, pectate lyases, pectoacetases, pectinases, pentosanases, perhydrolases, peroxidases, phenol oxidases, phosphatases, phospholipases, phytases, polygalacturonases, polyesterases, proteases, pullulanases, reductases, rhamnogalacturonases, beta-glucanases, tannase, transglutaminases, xylan acetyl esterases, xylanases, xyloglucanases, xylosidases, and any combination or mixture thereof.

14. The enzyme composition of embodiment 13, wherein the at least one additional enzyme is selected from the group consisting of: proteases, alpha-amylases, cellulases and mannanases.

Example 15A process for degrading a polyester or polyester-containing material, the process comprising

i) Contacting the polyester-containing material with a variant lipolytic enzyme as defined in any of examples 1-7 or a composition comprising a variant lipolytic enzyme as defined in examples 1-7, and optionally,

ii) rinsing the polyester-containing material.

Example 16. A process for enzymatic depolymerization of a polyester or polyester-containing material, the process comprising:

i) Contacting the polyester or polyester-containing material with a variant lipolytic enzyme as claimed in any of embodiments 1-7 or a composition comprising a variant lipolytic enzyme as claimed in embodiments 1-7 and optionally,

ii) recovering the monomers and/or oligomers of said polyester.

Embodiment 17. The method of embodiment 15 or 16 wherein the polyester is selected from the group consisting of: polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), polyethylene naphthalate (PEN), polyester polyurethane, poly (ethylene adipate) (PEA), and combinations thereof.

Examples

Example 1

Recombinant expression and production of Pseudomonas mendocina lipase variants

A codon-optimized synthetic gene (SEQ ID NO: 1) encoding a wild-type Pseudomonas mendocina lipase (SEQ ID NO: 2) was prepared and used as a template for constructing plasmids expressing variant polypeptides thereof. Lipase genes were produced by GeneArt AG (Regensburg, germany) or by Tewestery bioscience (san Francisco, USA) and cloned into pSB expression vectors using standard molecular biology techniques (Babe, L.M., et al (1998) Biotechnol Appl Biochem [ Biotechnology and applied biochemistry ] 27:117-24), to generate expression plasmids suitable for expression in Bacillus subtilis. The elements of the construct include: a DNA fragment comprising the aprE promoter sequence (SEQ ID NO: 3), a nucleotide sequence encoding the aprE signal peptide sequence (SEQ ID NO: 4) or the hybrid aprE-Menispermaceae lipase signal peptide sequence (SEQ ID NO: 5), a sequence corresponding to the gene encoding mature lipase, the BPN' terminator (SEQ ID NO: 6), and additional elements from pUB110 (McKenzie et al (1986) Plasmid [ Plasmid ] 15:93-103) (including replicase gene (reppUB), neomycin/kanamycin resistance gene (neo), bleomycin resistance marker (bleo)).

A suitable Bacillus subtilis host strain is transformed with the pSB expression plasmid using methods known in the art (WO 02/14490). The transformation mixture was plated onto LA plates containing 10ppm neomycin sulfate and incubated overnight at 37 ℃. Single colonies were picked and grown in Luria broth at 37℃under antibiotic selection.

To generate enzyme samples for screening, transformed bacillus subtilis cells were grown in medium (semi-defined medium enriched based on MOPS buffer, with urea as the primary nitrogen source, glucose as the primary carbon source, and supplemented with 1% soytone for robust cell growth) in each well of a 96-well microtiter plate (MTP, manufactured) at 37 ℃ for 68 hours. Cultures were harvested by centrifugation at 3600rpm for 15min and passed through a Miybag vacuum systemFilter board (EMD Milibo corporation, biller, mass., bill card (Biller)ica)) was filtered. The filtered culture supernatant was used in the following assay. Typically, the culture broth is diluted in 100mM Tris (pH 8) in 96 well plates (Neken Corp., NUNC), 267245). The enzyme concentration was determined by separating the protein fraction using a Zorbax 300SB-C3 column (Agilent) and running a linear gradient of 0.1% trifluoroacetic acid in water (buffer a) and 0.1% trifluoroacetic acid in acetonitrile (buffer B) and detection on a UHPLC at 220 nm. The enzyme concentration of the sample was calculated using the standard curve of the purified reference enzyme PEV 132.

Example 2

Enzymatic Activity of Pseudomonas mendocina Lipase variants

The enzyme activity of pseudomonas mendocina lipase variants (listed in table 1) was tested on PET (polyethylene terephthalate) substrates by measuring the hydrolysis of PET pellet substrates in solution. PET pellets were purchased from scientific polymer products company (Scientific Polymer Products) (catalog No. 138). One PET pellet (20-30 mg) was added to each well of a microtiter plate (can ken, 267245), and a detergent solution was added. In particular, the detergent solution consisted of two hundred microliters of formula A HDL (3.0 g/L) (composition in Table 2) prepared in 10mM Tris-HCl buffer (water hardness 6gpg, ca: mg=3:1, pH 8).

Plates without PET in a set of wells were also set up as controls for enzyme background. Twenty microliters of each enzyme sample was added to each well of the assay plate to initiate the reaction. The reaction was carried out at 40℃for 24 hours with shaking (180 rpm) in an incubation shaker (Inforsen Biotechnology Co., ltd. (Infors HT)). After incubation, 100ul of the reaction supernatant was transferred to a new UV transparent plate (Corning) 3635 and measured on a microplate reader (molecular instruments, spectraMax plus 384) at 240 nm. After subtracting the absorbance of the enzyme background plate, the resulting absorbance was taken as a measure of PET hydrolytic activity. Absorbance values are plotted against enzyme concentration. Each variant was assayed in triplicate. PET activity is reported as Performance Index (PI) values calculated by dividing the PET activity of each variant by the PET activity of the parent tested at the same protein concentration. Table 3 shows the polyesterase activity (performance index) of the variants from table 1 on PET substrates. Theoretical values of PET activity of the parent enzyme at the relevant protein concentration were calculated using parameters extracted from Langmuir (Langmuir) fit of the standard curve of the parent enzyme activity.

Example 3

Thermal stability of Pseudomonas mendocina variants

The stability of the pseudomonas mendocina lipase variants (shown in table 1) was tested under stress conditions in a 50% (v/v) aqueous solution of the HDL detergent of formulation a by measuring the residual activity of the sample after 16 hours incubation at 56 ℃. An aqueous solution of 67% (v/v) detergent was prepared and an enzyme sample from the filtered culture supernatant was mixed with the appropriate volume of the detergent solution to achieve a final detergent concentration of 50% (v/v). To measure initial (no stress) activity, an aliquot of this mixture was immediately diluted in 100mM Tris-HCl,0.1%Triton X-100 (pH 8) and activity was measured on the pNB substrate. A solution (1 mM) of the pNB substrate (4-nitrophenyl butyrate, sigma) was prepared by adding 0.2mL of a stock solution of pNB (100 mM in DMSO) to 20mL of buffer (100 mM Tris-HCl,0.1% Triton X-100, pH 8). Ten microliters of the diluted enzyme solution was mixed into 190ul of 1mM pNB in assay buffer in a 96 well plate (Costar, no. 9017, sameiser) to start the reaction. The plates were thoroughly mixed and absorbance at OD 405nm was monitored every 12 seconds for 3 minutes in a microplate reader (molecular instruments, spectromax plus 384). The Vmax value (in mOD/min) of the enzyme-free sample (blank) was subtracted from the Vmax value of the enzyme-containing sample. The resulting Vmax (in mOD/min) is recorded as the enzyme activity on the pNB substrate. Once the stress and non-stress activation values were measured by hydrolysis of pNB substrate as described above, the residual activity percentage (%) was calculated by taking the ratio of stress to non-stress activation and multiplying by 100. Table 4 shows the residual activity% of the tested pseudomonas mendocina lipase variants.

While the present disclosure has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, the present disclosure is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents, and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present disclosure. The section headings are not to be construed as necessarily limiting.

Sequence listing

SEQ ID NO. 1 (codon optimized Gene sequence of wild-type Lipase from Pseudomonas mendocina)

SEQ ID NO. 2 (amino acid sequence of wild-type lipase from Pseudomonas mendocina)

SEQ ID NO. 3 (aprE promoter DNA sequence)

SEQ ID NO. 4 (aprE Signal peptide DNA sequence)

SEQ ID NO. 5 (heterozygous aprE-Pseudomonas mendocina Lipase Signal peptide DNA sequence)

SEQ ID NO. 6 (BPN' terminator DNA sequence)

Claims

1. A variant lipolytic enzyme comprising an amino acid sequence having at least 70% identity to the full length amino acid sequence of SEQ ID No. 2, the variant lipolytic enzyme comprising the substitution T064V-T117L-T177N/R-I178L-F180P-Y182A-R190L-S205G-S212D-F226L-Y239I-L249P-S252I-L258F and further comprising at least one additional substitution selected from the group consisting of: V014S, R040A/T, G059Y, G061D, A066D, S070E, Q161H, G A/E, F207TL/T, V210I, Q227H, A236P, S244E, E Q and R256K, wherein said positions are numbered by reference to the amino acid sequence of SEQ ID NO:2 and wherein said variant has esterase activity.

2. The variant lipolytic enzyme of claim 1 wherein the variant comprises an amino acid sequence which is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the full length amino acid sequence of SEQ ID No. 2.

3. The variant lipolytic enzyme of claim 1 or 2, wherein the variant is derived from a parent enzyme comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the full-length amino acid sequence of SEQ ID No. 2.

4. The variant lipolytic enzyme of any of the preceding claims, wherein the variant comprises a combination of substitutions selected from the group consisting of: R40T-T64V-T117T-G175L-T180G-G175A-R190G-S205G-F207L-S212D-F226L-Y239I-L249P-S252I-L258F, R T-G61D-T64V-S70E-T117L-T177L-S178L-F180P-Y180A-R190L-S205G-F207T-S212D-F226L-Q227H-A236P-Y239I-L249P-S252I-E258Q-L F, R T64T-T64V-S70E-T117L-T117N-I178L-F182A-R182L-S205T-F207T-S212D-F226L-A236P-Y249P-S252I-E254Q-L5740A-T64V-E70T-T180F 180L-T180I-S182A-L182-D180F-L180; F226L-A236P-Y239I-L249P-S252I-E254Q-L258F, R T-T64V-S70E-T117L-Q161H-T177N-I178L-F180P-Y182A-R190L-S205G-F207T-S212D-F226L-A236P-Y239I-L249P-S252I-E254Q-L258F, R T-T64V-S70E-T117L-G117A-T177N-I178L 180P-Y182A-R190L-S205G-F207T-S212D-F226L-A236P-Y239I-L249P-S252P-E254Q-L258F, R T64V-S70E-T117L-T177N-I178L 180P-Y182A-R190L-S190G-S205T 210F 180P-E252A-F252D 252L-E258P-E252L-E252L-I254, R40T-T64V-T70E-T117L-T178L-F180P-Y182A-R190L-S205G-F207T-S212D-F226L-A236P-Y239I-S244E-L249P-S254I-E254Q-L258T 40T-T64V-S70E-T117L-T177N-I178L-F180P-Y182A-R190L-S205G-F207T-S212D-F226L-A236P-Y249P-S254Q-R256K-L258F, V A-G61Y-G61D-T64V-A66D-S70E-T117L-Q161H-T177R-I178L-F180P-Y180A-R190L-S205G-F207T-S210I-S212D-F226L-A236P-Y239I-L249P-S252I-E254Q-R256K-E254K-T64V-S70E-T117S-R40A-G59Y-G61D-T64V-S70E-R190L-S205G-F207T-V210I-S212D-F226L-A236P-Y239I-L249P-S252I-E254Q-R256K-L258 6240T-G61D-T64V-S70E-T117L-Q161H-T177R-178L-F180P-Y182A-R205G-F205T-V210I-S212D-F226L-A236P-Y239I-L249P-S252I-E254Q-R256K-L258F 258, and V14S-R40A-G59Y-G61D-T64V-A66D-S70E-T117L-Q161H-G175A-T177R-I178L-F180P-Y182A-R190L-S205G-F207T-V210I-S212D-F226L-A236P-Y239I-L249P-S252I-E254Q-R256K-L258F, wherein the positions are numbered by reference to the amino acid sequence of SEQ ID NO: 2.

5. The variant lipolytic enzyme of any of the preceding claims, wherein the variant has one or more improved properties when compared to a parent or reference lipolytic enzyme, wherein the improved properties are selected from improved stability, improved hydrolytic activity towards polyesters, or a combination thereof.

6. The variant lipolytic enzyme of any of the preceding claims, wherein the improved property is:

7. The lipolytic enzyme of any of claims 1-6 wherein the variant has hydrolytic activity on a polyester selected from the group consisting of: polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), polyethylene naphthalate (PEN), polyester polyurethane, poly (ethylene adipate) (PEA), and combinations thereof.

8. A polynucleotide comprising a nucleic acid sequence encoding the variant lipolytic enzyme of any of claims 1-7.

9. The polynucleotide of claim 8, wherein the nucleic acid sequence is operably linked to a promoter.

10. An expression vector or expression cassette comprising the polynucleotide of claim 8 or 9.

11. A recombinant host cell comprising the expression vector or expression cassette of claim 10.

12. An enzyme composition comprising the variant lipolytic enzyme of any of claims 1-7.

13. The enzyme composition of claim 12, wherein the composition further comprises at least one additional enzyme selected from the group consisting of: acyltransferases, alpha-amylases, beta-amylases, alpha-galactosidases, arabinosidases, aryl esterases, beta-galactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, endo-beta-1, 4-glucanases endo-beta-mannanase, esterase, exo-mannanase, feruloyl esterase, galactanase, glucoamylase, hemicellulase, enzyme preparation method, and pharmaceutical composition hexosaminidase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, mannanase metalloproteinases, nucleases (e.g., deoxyribonucleases and ribonucleases), oxidases, oxidoreductases, pectate lyases, pectoacetases, pectinases, pentosanases, perhydrolases, peroxidases, phenol oxidases, phosphatases, phospholipases, phytases, polygalacturonases, polyesterases, proteases, pullulanases, reductases, rhamnogalacturonases, beta-glucanases, tannase, transglutaminases, xylan acetyl esterases, xylanases, xyloglucanases, xylosidases, and any combination or mixture thereof.

14. The enzyme composition of claim 13, wherein the at least one additional enzyme is selected from the group consisting of: proteases, alpha-amylases, cellulases and mannanases.

15. A process for degrading a polyester or polyester-containing material, the process comprising

i) Contacting the polyester-containing material with a variant lipolytic enzyme according to any of claims 1-7 or a composition comprising a variant lipolytic enzyme according to any of claims 1-7 and optionally,

ii) rinsing the polyester-containing material.

16. A process for enzymatic depolymerization of a polyester or polyester-containing material, the process comprising:

i) Contacting the polyester or polyester-containing material with a variant lipolytic enzyme according to any of claims 1-7 or a composition comprising a variant lipolytic enzyme according to any of claims 1-7 and optionally,

ii) recovering the monomers and/or oligomers of said polyester.

17. The method of claim 15 or 16, wherein the polyester is selected from the group consisting of: polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), polyethylene naphthalate (PEN), polyester polyurethane, poly (ethylene adipate) (PEA), and combinations thereof.